Inhibitors of G protein-mediated signaling, methods of making them, and uses thereof

ABSTRACT

The invention provides compositions and methods useful for blocking G protein-mediated signaling events. Compositions provided by the invention relate to carboxy terminal Gα peptides, and minigenes which encode such peptides. The invention provides methods of making the minigenes, and methods of using either of the minigenes and the Gα carboxy terminal peptides encoded thereby, to block G protein-mediated signaling events. The invention also provides methods of using either of the minigenes and the Gα carboxy terminal peptides encoded thereby for the identification of unknown interactions between G proteins and G protein coupled receptors, and for the treatment of pathological disorders associated with G protein-mediated signaling events.

STATEMENT REGARDING FEDERALLY SUPPORTED RESEARCH OR DEVELOPMENT

[0001] This research was supported in part by U.S. Government funds(National Institutes of Health, grant numbers HL63341-01 and EI10291),and the U.S. Government may therefore have certain rights in theinvention.

BACKGROUND OF THE INVENTION

[0002] Many biologically active molecules transduce their signalsthrough heptahelical receptors called G protein coupled receptors(GPCRs), which interact specifically with heterotrimeric guaninenucleotide-binding proteins, called G proteins. The interaction of a Gprotein with its cognate GPCR results in the initiation of intracellularsignaling events which, in turn, lead to a variety of important cellularresponses (Hamm et al., 1996, Curr. Opin. Cell. Biol. 8: 189-196; Hamm,1998, Science 273: 669-672). Stimulation of a GPCR by its appropriateagonist causes conformational changes within the receptor that lead tothe interaction of the activated receptor with a specific heterotrimericG protein. Thus, the GPCR-G protein interaction plays an important rolein determining the specificity and temporal characteristics of a varietyof cellular responses.

[0003] Heterotrimeric G proteins are composed of a single α subunitcomplexed with the βγ dimer. Molecular cloning has resulted in theidentification of 18 distinct α subunits, 5 β subunits, and 12 γsubunits. G proteins are usually divided into four subfamilies G_(i),G_(s), G_(q), and G₁₂ based on the sequence similarity of the Gαsubunit. Several lines of evidence suggest that the interaction betweena given GPCR and its cognate G protein involves multiple sites ofcontact on both proteins. All three intracellular loops as well as thecarboxyl terminal tail of the receptor have been implicated. The GPCR isthought to interact with all three subunits of the G protein. As thereceptor-G protein interaction can be disrupted by a number oftreatments that block the carboxyl terminus, including pertussistoxin-catalyzed ADP-ribosylation of G_(i) and binding of monoclonalantibodies, the carboxy terminal region of the Gα subunit has been themost intensely investigated contact site. These studies have shown thatthe Gα carboxy terminal region is important not only to the interaction,but also plays a critical role in defining receptor specificity (Hamm etal., 1988, Science 241: 832-5; Osawa et al., 1995, J. Biol. Chem. 270:31052-8; Garcia et al., 1995, EMBO 14: 4460-9; Sullivan et al., 1987,Nature 330: 758-760; Rasenick et al., 1994, J. Biol. Chem. 269:21519-21525; West et al., 1985, J. Biol. Chem. 260: 14428-30; Conklin etal., 1993, Nature 363: 274-276; Conklin et al., 1996, Mol. Pharmacol.50: 885-890). Furthermore, it has been shown that peptides correspondingto the carboxy terminal region of a Gα subunit can block GPCR signalingevents (Hamm et al., 1988, Science 241: 832-5; Gilchrist et al., 1998,J. Biol. Chem 273: 14912-19).

[0004] Because many medically significant biological processes aremediated by G proteins and their downstream effector molecules, theGPCRs with which they interact, have been the focus of intense drugdiscovery efforts (Holler et al., 1999, Cell. Mol. Life Sci. 55: 257-70;Farfel et al., 1999, New Engl. J. Med. 340: 1012-20). A number oftherapeutic agents targeting GPCRs have been discovered. Traditionally,the agonist binding site on the GPCR is the point of intervention.However, for some GPCR's, classical antagonists have been difficult toidentify. Such is the case for proteinase activated receptors (PAR),classical antagonists are ineffective due to the unique mechanism ofenzymatic cleavage of the receptor and generation of a tethered ligand.In other cases, intrinsic or constitutive activity of receptors directlyleads to pathology. Thus, alternative targets for blocking downstreamconsequences of GPCR signaling are needed. Agents are needed that caninhibit GPCR by blocking the receptor-G protein interface. The presentinvention satisfies these needs.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention includes an isolated nucleic acidcomprising a minigene, wherein said minigene encodes a modified carboxyterminal Gα peptide, wherein said peptide blocks the site of interactionbetween a G protein and a G protein coupled receptor in a cell, such asa human cell. In addition, the minigene can further comprise one or moreof a promoter, a ribosomal binding site, a translation initiation codon,and a translation termination codon.

[0006] In one embodiment, the nucleotide sequence of a minigene of theinvention can be one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, and 14.

[0007] In another embodiment, a minigene encodes a modified carboxyterminal Gα peptide having one of the following general formulas: MGX,MX, and MZX,

[0008] wherein M is a methionine amino acid residue,

[0009] wherein G is a glycine amino acid residue,

[0010] wherein Z is an amino acid residue other than a glycine aminoacid residue, and

[0011] wherein X is a carboxy terminal Gα peptide which comprises anamino acid sequence of the carboxy terminus of a Gα subunit, and has theproperty of binding a G protein coupled receptor. In this embodiment, Xcan comprise from at least about three contiguous amino acids to atleast about 54 contiguous amino acids, from at least about threecontiguous amino acids to at least about eleven contiguous amino acids,and at least about eleven contiguous amino acids. In one embodiment, Xcomprises the seven contiguous terminal amino acid residues of thecarboxy terminus of a Gα subunit. For example, the amino acid sequenceof a modified carboxy terminal Gα peptide can be one of SEQ ID NOs: 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29.

[0012] The present invention also includes a composition comprising amodified carboxy terminal Gα peptide having a general formula selectedfrom the group consisting of MGX, MX, and MZX,

[0013] wherein M is a methionine amino acid residue,

[0014] wherein G is a glycine amino acid residue,

[0015] wherein Z is an amino acid residue other than a glycine aminoacid residue, and

[0016] wherein X is a carboxy terminal Gα peptide which comprises anamino acid sequence of the carboxy terminus of a Gα subunit, and has theproperty of binding a G protein coupled receptor. In this embodiment, Xcan comprise from at least about three contiguous amino acids to atleast about 54 contiguous amino acids, from at least about threecontiguous amino acids to at least about eleven contiguous amino acids,and at least about eleven contiguous amino acids. In one embodiment, Xcomprises the seven contiguous terminal amino acid residues of thecarboxy terminus of a Gα subunit. For example, the amino acid sequenceof a modified carboxy terminal Gα peptide can be one of SEQ ID NOs: 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29.

[0017] Additionally, the invention includes a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and one of a modifiedcarboxy terminal Gα peptide and a minigene encoding a modified carboxyterminal Gα peptide.

[0018] In one aspect, the invention encompasses a method of inhibiting aG protein-mediated signaling event in a cell. This method comprisesadministering to a cell, preferably a human cell, one of a modifiedcarboxy terminal Gα peptide, and an isolated nucleic acid comprising aminigene which encodes a modified carboxy terminal Gα peptide, wherebyfollowing the administration, the carboxy terminal Gα peptide inhibitsthe G protein mediated signaling event in the cell.

[0019] Additionally, the invention includes a method of blocking thesite of interaction between a G protein and a G protein coupled receptorin a cell. This method comprises administering to a cell, preferably ahuman cell, one of a modified carboxy terminal Gα peptide, and anisolated nucleic acid comprising a minigene which encodes a modifiedcarboxy terminal Gα peptide, whereby following the administration, themodified carboxy terminal Gα peptide blocks the site of interactionbetween the G protein and the G protein coupled receptor in the cell.

[0020] Further, the invention includes a method of inhibiting one ormore of migration, permeability, and proliferation of a cell. Thismethod comprises administering to a cell, preferably a human cell, oneof a modified carboxy terminal Gα peptide, and an isolated nucleic acidcomprising a minigene which encodes a modified carboxy terminal Gαpeptide, wherein the modified carboxy terminal Gα peptide blocks a Gprotein-mediated signaling event in a cell, thereby inhibiting one ormore of migration, permeability, and proliferation of a cell.

[0021] In one embodiment, the preferred cell is a human cell.

[0022] In other embodiments, the modified carboxy terminal Gα peptide,either as administered or as expressed from a minigene of the invention,has a general formula selected from the group consisting of MGX, MX, andMZX,

[0023] wherein M is a methionine amino acid residue,

[0024] wherein G is a glycine amino acid residue,

[0025] wherein Z is an amino acid residue other than a glycine aminoacid residue, and

[0026] wherein X is a carboxy terminal Gα peptide comprising an aminoacid sequence of the carboxy terminus of a Gα subunit, and having theproperty of binding a G protein coupled receptor. In these embodiments,X can comprise from at least about three contiguous amino acids to atleast about 54 contiguous amino acids, from at least about threecontiguous amino acids to at least about eleven contiguous amino acids,and at least about eleven contiguous amino acids. In one embodiment, Xcomprises the seven contiguous terminal amino acid residues of thecarboxy terminus of a Gα subunit. For example, the amino acid sequenceof a modified carboxy terminal Gα peptide can be one of SEQ ID NOs: 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29. As analternative example, the nucleotide sequence of a minigene can be any ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,and 14.

[0027] The invention encompasses a method of identifying which G proteinbinds a G protein coupled receptor in a cell. This method comprisesadministering to the cell an isolated nucleic acid comprising a minigenewhich encodes a modified carboxy terminal Gα peptide, and assessing thelevel of occurrence of a signaling event in the cell. According to thismethod, the signaling event is associated with the G protein coupledreceptor, and a measurable reduction in the level of occurrence of thesignaling event in the cell compared with the level of occurrence of thesame event in a cell to which the isolated nucleic acid is notadministered, is an indication that said modified carboxy terminal Gαpeptide identifies a G protein that binds said G protein coupledreceptor.

[0028] The invention additionally includes a method of treating apathological disorder in a mammal. This method comprises administeringto the mammal an amount of either a modified carboxy terminal Gα peptideor an isolated nucleic acid comprising a minigene which encodes amodified carboxy terminal Gα peptide. According to the method, theamount of either the modified carboxy terminal Gα peptide or theisolated nucleic acid is sufficient to inhibit a G protein-mediatedsignaling event associated with the pathological disorder, and therebyalleviate at least one symptom of the pathological disorder.

[0029] In one embodiment, the method of treating a pathological disorderin a mammal includes administering to the mammal a modified carboxyterminal Gα peptide having a general formula selected from the groupconsisting of MGX, MX, and MZX,

[0030] wherein M is a methionine amino acid residue,

[0031] wherein G is a glycine amino acid residue,

[0032] wherein Z is an amino acid residue other than a glycine aminoacid residue, and

[0033] wherein X is a carboxy terminal Gα peptide which comprises anamino acid sequence of the carboxy terminus of a Gα subunit, and has theproperty of binding a G protein coupled receptor. In these embodiments,X can comprise from at least about three contiguous amino acids to atleast about 54 contiguous amino acids, from at least about threecontiguous amino acids to at least about eleven contiguous amino acids,and at least about eleven contiguous amino acids. In one embodiment, Xcomprises the seven contiguous terminal amino acid residues of thecarboxy terminus of a Gα subunit. For example, the amino acid sequenceof a modified carboxy terminal Gα peptide can be one of SEQ ID NOs: 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29. As analternative example, the nucleotide sequence of a minigene can be any ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,and 14.

[0034] In multiple embodiments, the method of the invention is usefulfor treating a pathological disorder selected from the group consistingof stroke, myocardial infarction, restenosis, atherosclerosis,hypotension, hypertension, angina pectoris, acute heart failure,cardiomyocyte apoptosis, cancers, bacterial infections, fungalinfections, protozoan infections, viral infections, septic shock, pain,chronic allergic disorders, asthma, inflammatory bowel disease,osteoporosis, rheumatoid arthritis, Grave's disease, post-operativeileus, diabetes, adult respiratory distress syndrome, myastenia gravis,cardiovascular disease, congestive heart failure, Chagas disease,disorders associated with solid organ transplant, vascular sclerosis,chronic rejection, chronic obstructive pulmonary disease, urinaryretention, testotoxicosis, infertility, ulcers, obesity, benignprostatic hypertrophy, anxiety, epilepsy, schizophrenia, manicdepression, Parkinson's disease, Alzheimer's disease, delirium,dementia, drug addiction, anorexia, and bulimia.

[0035] In another aspect, the invention includes a method of treating adisorder associated with an endothelial cell in a mammal, comprisingadministering to the endothelial cell of the mammal an isolated nucleicacid comprising a minigene, wherein said minigene encodes a modifiedcarboxy terminal Gα peptide, or a modified carboxy terminal Gα peptideencoded thereby. According to this method, the peptide blocks a Gprotein-mediated signaling event in the endothelial cell, and therebyalleviates at least one symptom of the disorder associated with theendothelial cell in the mammal.

[0036] The invention additionally includes a composition comprising apeptide having an amino acid sequence selected from the group consistingof SEQ ID Nos: 39, 40, 41, 42, 43, 44, 45, 46, and 47.

[0037] Further, the invention includes a method of preventing conceptionin a female mammal. This method comprises administering to the femalemammal either of a modified carboxy terminal Gα peptide or an isolatednucleic acid comprising a minigene, wherein the minigene encodes amodified carboxy terminal Gα peptide, thereby preventing conception inthe female mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1 is a diagram which depicts the design of a typical minigeneand its insertion into the expression vector, pcDNA 3.1 (Invitrogen;Carlsbad, Calif.).

[0039]FIG. 2, comprising FIGS. 2A, 2B, and 2C, depicts the nucleotidesequences of representative minigenes, amino acid sequences of peptidesencoded thereby, and amino acid sequences of representative Gα carboxyterminal peptides. FIG. 2A depicts the nucleotide sequences (SEQ ID NOs1-14) of representative minigenes encoding Gα carboxy terminal peptidesof the general formula, MGX, wherein X is an amino acid sequencecorresponding the carboxy terminal region of a human Gα subunit. Theportion of the nucleotide sequence depicted for each minigene includes aBamHI restriction endonuclease site, a ribosomal binding sequence, thetranslation initiation codon, methionine, a glycine codon, thenucleotide sequence encoding the Gα carboxy terminal peptide sequence of11 amino acids, and the translation stop codon immediately downstream ofthe sequence encoding the peptide. FIG. 2B depicts the amino acidsequences (SEQ ID NOs. 15-29) of Gα carboxy terminal peptides encoded byrepresentative minigenes. These peptides are of the general formula,MGX, wherein X is an amino acid sequence corresponding to the carboxyterminal region of a Gα subunit. FIG. 2C depicts the nucleotidesequences (SEQ ID NOs. 30-38) encoding the carboxy terminal regions ofselected human Gα subunit proteins. FIG. 2D depicts the amino acidsequences (SEQ ID NOs. 39-47) of peptides corresponding to the carboxyterminal region of selected human Gα subunit proteins.

[0040]FIG. 3, comprising FIGS. 3A and 3B, depicts analyses of minigeneexpression in transiently transfected HEK 293 cells. FIG. 3A is an imageof a gel which depicts the separation of DNA fragments generated in areverse transcription-polymerase chain reaction (RT-PCR) on a 1.5%agarose gel. FIG. 3B is a series of graphs which depicts thechromatograms resulting from an analysis of cytosolic extracts usinghigh pressure liquid chromatography (HPLC).

[0041]FIG. 4, comprising FIGS. 4A, 4B, and 4C, is a series of graphswhich depicts a comparison of the acetylcholine (Ach) activation ofinwardly rectifying potassium channels (GIRKs) in HEK 293 cellstransiently transfected with selected minigenes.

[0042]FIG. 5 is a graph which depicts a comparison of the level ofcyclic adenosine monophosphate (cAMP) accumulation in humanmicrovascular endothelial cells (HMEC) transiently transfected withpcDNA 3.1, (vector only), pcDNA-Gα_(i1/2) (Gi1/2), or pcDNA-Gα_(i)R(GiR). The cells are stimulated with either isoproterenol or bothisoproterenol and thrombin. Control cells are not stimulated.

[0043]FIG. 6 is a graph which depicts a comparison of thethrombin-stimulated proliferative response of bovine pulmonary arterialendothelial (BPAE) cells transiently transfected with pcDNA 3.1,(pcDNA), pcDNA-Gα_(i)R (GiR), or pcDNA-Gα_(i1/2) (Gi_(1/2)). The datarepresents at least three independent experiments, each done induplicate.

[0044]FIG. 7 is a graph which depicts a comparison ofthrombin-stimulated luciferase acivity as a measure of PAR-1 geneexpression in HMEC which are either treated with pertussis toxin ortransiently transfected with pcDNA 3.1, (pcDNA3.1(−)), pcDNA-Gα_(i1/2)(Gi), or pcDNA-Gα_(i)R (Gi Random).

[0045]FIG. 8 is a graph which depicts a comparison of the level ofthrombin-induced intracellular Ca²⁺ rise in HMEC transiently transfectedwith pcDNA-Gα_(i)R (GiR), pcDNA-Gα_(i1/2) (Gi1/2), or pcDNA-Gα_(q) (Gq).

[0046]FIG. 9 is a graph which depicts a comparison of the level ofinositol phosphate (IP) accumulation in HMEC transiently transfectedwith pcDNA 3.1, (pcDNA), pcDNA-Gα_(i)R (GiR), pcDNA-Gα_(i1/2) (Gi1/2),or pcDNA-Gα_(q) (Gq). The data represent at least three independentexperiments performed in triplicate and expressed as a mean±standarderror of the mean (SEM).

[0047]FIG. 10 is a graph which depicts a comparision of the level ofthrombin-induced, mitogen-activated protein kinase (MAPK) activity inHMEC transiently transfected with selected minigenes.

[0048]FIG. 11, comprising FIGS. 11A-D, is group of photographs depictingthrombin-induced stress-fiber formation in HMEC transiently transfectedwith pcDNA 3.1, pcDNA-Gα₁₂, or pcDNA-Gα₁₃.

[0049]FIG. 12 is a graph depicting-thrombin-induced adhesion of HL60cells to HMEC transiently transfected with pcDNA 3.1, pcDNA-Gα_(i)R,pcDNA-Gα₁₂, or pcDNA-Gα₁₃.

[0050]FIG. 13 is a graph which depicts a comparison of the level ofcyclic adenosine monophophate (cAMP) accumulation in HMEC transientlytransfected with pcDNA 3.1, pcDNA-Gα_(s), or pcDNA-Gα_(i)R. The cellsare stimulated with isoproterenol which generates cAMP via activation ofβ-adrenergic receptors.

[0051]FIG. 14 is a graph which depicts a comparison of the relativelevels of thrombin-induced transendothelial electrical resistance (TEER)in HMEC transiently transfected with selected minigenes.

DETAILED DESCRIPTION OF THE INVENTION

[0052] The present invention encompasses compositions and methods usefulfor blocking G protein-mediated signaling events. The invention stemsfrom the discovery that a peptide corresponding to the carboxy terminalregion of a G protein a subunit (Gα) can interfere with the interactionbetween the G protein and its cognate receptor (GPCR), and thereby blocksubsequent signaling by the GPCR.

[0053] The present invention relates to carboxy terminal Gα peptides,and nucleotide sequences and minigenes which encode such peptides. Theinvention further relates to methods of making Gα peptide-encodingminigenes, and methods of using minigenes and the Gα carboxy terminalpeptides encoded thereby, to block. G protein-mediated signaling events.The invention additionally includes methods of using minigenes and theGα carboxy terminal peptides encoded thereby for the identification ofas yet unknown GPCR-G protein interactions, and for the treatment ofpathological disorders associated with G protein-mediated signalingevents.

[0054] Definitions

[0055] As used herein, each of the following terms has the meaningassociated with it in this section.

[0056] The articles “a” and “an” are used herein to refer to one or tomore than one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

[0057] As used herein, amino acids are represented by the full namethereof, by the three letter code corresponding thereto, or by theone-letter code corresponding thereto, as indicated in the following:Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp D GlutamicAcid Glu E Lysine Lys K Arginine Arg R Histidine His H Tyrosine Tyr YCysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser S ThreonineThr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu L IsoleucineIle I Methionine Met M Proline Pro P Phenylalanine Phe F Tryptophan TrpW

[0058] “Amplification” refers to any means by which a polynucleotidesequence is copied and thus expanded into a larger number ofpolynucleotide molecules, for example, by reverse transcription,polymerase chain reaction, or ligase chain reaction.

[0059] A “coding region” of a gene consists of the nucleotide residuesof the coding strand of the gene and the nucleotides of the non-codingstrand of the gene which are homologous with or complementary to,respectively, the coding region of an mRNA molecule which is produced bytranscription of the gene.

[0060] “Encoding” refers to the inherent property of specific sequencesof nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA,to serve as templates for synthesis of other polymers and macromoleculesin biological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

[0061] Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence.

[0062] Oligonucleotides which contain at least one phosphorothioatemodification are known to confer upon the oligonucleotide enhancedresistance to nucleases. Specific examples of modified oligonucleotidesinclude those which contain phosphorothioate, phosphotriester, methylphosphonate, short chain alkyl or cycloalkyl intersugar linkages, orshort chain heteroatomic or heterocyclic intersugar (“backbone”)linkages. In addition, oligonucleotides having morpholino backbonestructures (U.S. Pat. No. 5,034,506) or polyamide backbone structures(Nielsen et al., 1991, Science 254: 1497) may also be used.“Complementary” refers to the broad concept of sequence complementaritybetween regions of two nucleic acid strands or between two regions ofthe same nucleic acid strand. It is known that an adenine residue of afirst nucleic acid region is capable of forming specific hydrogen bonds(“base pairing”) with a residue of a second nucleic acid region which isantiparallel to the first region if the residue is thymine or uracil.Similarly, it is known that a cytosine residue of a first nucleic acidstrand is capable of base pairing with a residue of a second nucleicacid strand which is antiparallel to the first strand if the residue isguanine. A first region of a nucleic acid is complementary to a secondregion of the same or a different nucleic acid if, when the two regionsare arranged in an antiparallel fashion, at least one nucleotide residueof the first region is capable of base pairing with a residue of thesecond region. Preferably, the first region comprises a first portionand the second region comprises a second portion, whereby, when thefirst and second portions are arranged in an antiparallel fashion, atleast about 50%, and preferably at least about 75%, at least about 90%,or at least about 95% of the nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion. More preferably, all nucleotide residues of the first portionare capable of base pairing with nucleotide residues in the secondportion.

[0063] As used herein, the term “adjacent” is used to refer tonucleotide sequences which are directly attached to one another, havingno intervening nucleotides. By way of example, the pentanucleotide5′-AAAAA-3′ is adjacent the trinucleotide 5′-TTT-3′ when the two areconnected thus: 5′-AAAAATTT-3′ or 5′-TTTAAAAA-3′, but not when the twoare connected thus: 5′-AAAAACTTT-3′.

[0064] A first region of an oligonucleotide “flanks” a second region ofthe oligonucleotide if the two regions are adjacent one another or ifthe two regions are separated by no more than about 1000 nucleotideresidues, and preferably no more than about 100 nucleotide residues.

[0065] “Homologous” as used herein, refers to the subunit sequencesimilarity between two polymeric molecules, e.g., between two nucleicacid molecules, e.g., two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′TATGGC share 50%homology. As further example, a “homolog” of a carboxy terminal Gαpeptide is a peptide that is homologous in sequence, as defined herein,to a carboxy terminal Gα peptide.

[0066] As used herein, “homology” is used synonymously with “identity.”Percent identity of one polynucleotide or polypeptide with respect toanother polynucleotide or polypeptide may be determined using anyavailable algorithm, such as the BLAST program.

[0067] An “isolated nucleic acid” refers to a nucleic acid segment orfragment which has been separated from sequences which flank it in anaturally occurring state, e.g., a DNA fragment which has been removedfrom the sequences which are normally adjacent to the fragment, e.g.,the sequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g, asa cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

[0068] A “ligand” is a compound that specifically binds to a targetreceptor.

[0069] A “receptor” is a compound that specifically binds to a ligand.

[0070] In the context of the present invention, the followingabbreviations for the commonly occurring nucleic acid bases are used.“A” refers to adenosine, “C” refers to cytidine, “G” refers toguanosine, “T” refers to thymidine, and “U” refers to uridine.

[0071] By describing two polynucleotides as “operably linked” is meantthat a single-stranded or double-stranded nucleic acid moiety comprisesthe two polynucleotides arranged within the nucleic acid moiety in sucha manner that at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked to the coding region of a gene isable to promote transcription of the coding region.

[0072] A “polynucleotide” means a single strand or parallel andanti-parallel strands of a nucleic acid. Thus, a polynucleotide may beeither a single-stranded or a double-stranded nucleic acid.

[0073] The term “nucleic acid” typically refers to largepolynucleotides.

[0074] The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 100 nucleotides. Itwill be understood that when a nucleotide sequence is represented by aDNA sequence (i.e., A, T, G, C), this also includes an RNA sequence(i.e., A, U, G, C) in which “U” replaces “T.”

[0075] Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

[0076] The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences” or as being“upstream of” of the reference point; sequences on the DNA strand whichare 3′ to a reference point on the DNA are referred to as “downstreamsequences” or as being “downstream of” the reference point.

[0077] A “portion” of a polynucleotide means at least about twentysequential nucleotide residues of the polynucleotide. It is understoodthat a portion of a polynucleotide may include every nucleotide residueof the polynucleotide.

[0078] “Primer” refers to a polynucleotide that is capable ofspecifically hybridizing to a designated polynucleotide template andproviding a point of initiation for synthesis of a complementarypolynucleotide. Such synthesis occurs when the polynucleotide primer isplaced under conditions in which synthesis is induced, i.e., in thepresence of nucleotides, a complementary polynucleotide template, and anagent for polymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

[0079] “Recombinant polynucleotide” refers to a polynucleotide havingsequences that are not naturally joined together. An amplified orassembled recombinant polynucleotide may be included in a suitablevector, and the vector can be used to transform a suitable host cell. Arecombinant polynucleotide may serve a non-coding function (e.g.,promoter, origin of replication, ribosome-binding site, etc.) as well.

[0080] As used herein, the term “promoter sequence” means a nucleic acidsequence which is required for expression of a gene product operablylinked to the promoter sequence. In some instances, this sequence may bethe core promoter sequence and in other instances, this sequence mayalso include an enhancer sequence and other regulatory elements whichare required for expression of the gene product. The promoter sequencemay, for example, be one which expresses the gene product in a tissuespecific manner.

[0081] A “constitutive” promoter is one which is actively transcribed ina cell independent of the addition of an exogenous inducer agent.Similarly, a “constitutively activated” G protein receptor is a receptorwhich is actively initiating G protein-mediated signaling eventsindependent of the addition of an exogenous inducing or stimulatingagent As used herein, the term “reporter gene” means a gene, theexpression of which can be detected using a known method. By way ofexample, the Escherichia coli lacZ gene may be used as a reporter genein a medium because expression of the lacZ gene can be detected usingknown methods by adding the chromogenic substrateo-nitrophenyl-β-galactoside to the medium (Gerhardt et al., eds., 1994,Methods for General and Molecular Bacteriology, American Society forMicrobiology, Washington, D.C., p. 574).

[0082] A “restriction endonuclease site” is a portion of adouble-stranded nucleic acid which is recognized by a restrictionendonuclease.

[0083] A portion of a double-stranded nucleic acid is “recognized” by arestriction endonuclease if the endonuclease is capable of cleaving bothstrands of the nucleic acid at the portion when the nucleic acid and theendonuclease are contacted.

[0084] By the term “specifically binds,” as used herein, is meant acompound, e.g., a protein, a nucleic acid, an antibody, and the like,which recognizes and binds a specific molecule, but does notsubstantially recognize or bind other molecules of a similar type. Byway of example, a G protein specifically binds a GPCR if the G proteinsubstantially binds only that GPCR, and does not substantially bindother membrane receptors.

[0085] Conventional notation is used herein to refer to polypeptide andpeptide amino acid sequences: the amino terminus of an amino acidsequence is the 5′-end; the the carboxy terminus of an amino acidsequence is the 3′-end. Amino acid sequences are listed herein from the5′ end to the 3′ end of the sequence, as read left to right.

[0086] The terms “Gα carboxy terminal peptide” and “carboxy terminal Gαpeptide” are used interchangeably herein. The terns “carboxy terminus”and “carboxy terminal region” of a “Gα subunit” or an “a subunitprotein” are used herein to refer to the 3′ end of an α subunit of a Gprotein. The sequence of “seven contiguous terminal amino acid residues”of a Gα subunit indicates the sequence of the seven contiguous aminoacids at the 3′ end of the Gα subunit amino acid sequence. By way ofexample, in a Gα subunit with the amino acid sequence of IKENLKDCGLF atthe 3′ end, the sequence of the seven contiguous terminal amino acidresidues is -LKDCGLF.

[0087] Peptides may incorporate amino acid residues which are modifiedwithout affecting activity. For example, the termini may be derivatizedto include blocking groups, i.e. chemical substituents suitable toprotect and/or stabilize the N- and C-termini from “undesirabledegradation”, a term meant to encompass any type of enzymatic, chemicalor biochemical breakdown of the compound at its termini which is likelyto affect the function of the compound as an anti-inflammatory agent,i.e. sequential degradation of the compound at a terminal end thereof.

[0088] Blocking groups include protecting groups conventionally used inthe art of peptide chemistry which will not adversely affect the in vivoactivities of the peptide. For example, suitable N-terminal blockinggroups can be introduced by alkylation or acylation of the N-terminus.Examples of suitable N-terminal blocking groups include C₁-C₅ branchedor unbranched alkyl groups, acyl groups such as formyl and acetylgroups, as well as substituted forms thereof, such as theacetamidomethyl (Acm) group. Desamino analogs of amino acids are alsouseful N-terminal blocking groups, and can either be coupled to theN-terminus of the peptide or used in place of the N-terminal reside.Suitable C-terminal blocking gruops, in which the carboxyl group of theC-terminus is either incorporated or not, include esters, ketones oramides. Ester or ketone-forming alkyl groups, particularly lower alkylgroups such as methyl, ethyl and propyl, and amide-forming amino groupssuch as primary amines (—NH₂), and mono- and di-alkylamino groups suchas methylamino, ethylamino, dimethylamino, diethylamino,methylethylamino and the like are examples of C-terminal blockinggroups. Descarboxylated amino acide analogues such as agmatine are alsouseful C-terminal blocking groups and can be either coupled to thepeptide's C-terminal residue or used in place of it. Further, it will beappreciated that the free amino and carboxyl groups at the termini canbe removed altogether from the peptide to yield desamino anddescarboxylated forms thereof without affect on peptide activity.

[0089] Other modifications can also be incorporated without adverselyaffecting anti-inflammatory activity and these include, but are notlimited to, substitution of one or more of the amino acids in thenatural L-isomeric form with amino acids in the D-isomeric form. Thus,the peptide may include one or more D-amino acide resides, or maycomprise amino acids which are all in the D-form. Retro-inverso forms ofpeptides in accordance with the present invention are also contemplated,for example, inverted peptides in whicch all amino acids are substitutedwith D-amino acid forms.

[0090] Acid addition salts of the present invention are alsocontemplated as functional equivalents. Thus, a peptide in accordancewith the present invention treated with an inorganic acid such ashydrochloric, hydrobromic, sulfuric, nitice, phosphoric, and the like,or an organic acid such as an acetic, propionic, glycolic, pyruvic,oxalic, malic, malonic, succinic, maleic, fumaric, tataric, citric,benzoic, cinnamie, mandelic, methanesulfonic, ethanesulfonic,p-toluenesulfonic, salicyclic and the like, to provide a water solublesalt of the peptide is suitable for use as an anti-inflammatory agent.

[0091] A “vector” is a composition of matter which comprises an isolatednucleic acid and which can be used to deliver the isolated nucleic acidto the interior of a cell. Numerous vectors are known in the artincluding, but not limited to, linear polynucleotides, polynucleotidesassociated with ionic or amphiphilic compounds, plasmids, and viruses.Thus, the term “vector” includes an autonomously replicating plasmid ora virus. The term should also be construed to include non-plasmid andnon-viral compounds which facilitate transfer of nucleic acid intocells, such as, for example, polylysine compounds, liposomes, and thelike. Examples of viral vectors include, but are not limited to,adenoviral vectors, adeno-associated virus vectors, retroviral vectors,and the like.

[0092] “Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses that incorporate the recombinant polynucleotide.

[0093] As used herein, “alleviating” a pathological disorder meansreducing the severity of the symptoms of the pathological disorder.

[0094] As used herein, to “treat” means reducing the frequency withwhich symptoms of a pathological disorder are experienced by a mammalhaving the disorder.

[0095] Description

[0096] The present invention relates to an isolated nucleic acidcomprising a minigene, wherein the minigene encodes a modified carboxyterminal Gα peptide. As exemplified in FIG. 1, a typical minigene of theinvention can further comprise one or more of the following elements: apromoter sequence, a ribosome-binding nucleotide sequence, a methioninecodon for translation initiation, an optional amino acid codon forstabilization of the translated peptide, a stop codon for translationtermination, and a restriction endonuclease site. Alternatively, aminigene described herein can be incorporated into a vector, such as anexpression vector, adjacent to one or more of a promoter sequence, aribosome-binding nucleotide sequence, a methionine codon for translationinitiation, an optional amino acid codon for stabilization of thetranslated peptide, a stop codon for translation termination, and arestriction endonuclease site.

[0097] The polynucleotide comprising the promotor sequence can be anypolynucleotide comprising a promoter sequence, for example, and withoutlimitation, the polynucleotide comprising the promoter sequence can beany viral-, bacterial-, yeast-, drosophila-, or mammalian-derivedpromoter sequence that is either known or becomes known, which is usefulin the minigene of the invention. Suitable promoter sequences caninclude, but are not limited to, a T7 promotor, an Sp6 promotor, acytomegalovirus (CMV) promotor, and a β-actin promotor.

[0098] The polynucleotide comprising the ribosome binding site may beany polynucleotide comprising ribosome binding site, for example, andwithout limitation, the polynucleotide comprising the ribosome bindingsite can be any viral-, bacterial-, yeast- or mammalian-derived ribosomebinding site that is either known or becomes known, which is useful inthe minigene of the invention.

[0099] In one embodiment, the minigene of the invention comprises aminigene sequence encoding a modified carboxy terminal Gα peptide havingone of the following general formulas: MGX, MZX, or MX, wherein M is amethionine amino acid residue, G is a glycine amino acid residue, Z isan amino acid residue other than glycine, and X is a carboxy terminal Gαpeptide sequence. By way of example, a minigene sequence of theinvention can be any of the nucleotide sequences depicted in FIG. 2A(SEQ ID NOs: 1-14) which encode the peptide sequences depicted in FIG.2B (SEQ ID Nos: 15-29). By way of further example, a minigene of theinvention can comprise a nucleotide sequence, such as a sequencedepicted in FIG. 2C (SEQ ID Nos: 30-38), which encodes a modifiedcarboxy terminal Gα peptide having the formula MIX or MX, wherein I isan isoleucine amino acid residue and X is an amino acid sequence such asany one of SEQ ID Nos 39-47. In this embodiment, the modified carboxyterminal Gα peptide encoded by a minigene of the invention can comprisean amino acid sequence of the carboxy terminus of any Gα subunit whichis known or becomes known, and can be any such peptide which exhibitsthe property of binding a G protein coupled receptor and is useful inthe methods of the invention.

[0100] Included in the present invention is a minigene encoding amodified carboxy terminal Gα peptide comprising from at least aboutthree contiguous amino acids to at least about 54 contiguous aminoacids, and from at least about three contiguous amino acids to at leastabout eleven contiguous amino acids. By way of example, a preferredminigene can encode a modified peptide comprising at least about 13contiguous amino acids such as any of the peptides depicted in FIG. 2B(SEQ ID Nos: 15-29) or FIG. 2D (SEQ ID Nos: 39-47). Nucleotide sequencesof exemplary minigenes are depicted in FIG. 2A (SEQ ID Nos: 1-14).

[0101] A minigene of the invention should not be construed to be limitedsolely to encoding a carboxy terminal Gα peptide disclosed herein.Rather, the invention should be construed to include a minigene whichencodes any amino acid sequence that shares substantial homology withthe amino acid sequence of a Gα carboxy terminal peptide sequence whichis known or becomes known. For example, the invention can include aminigene which encodes a modified carboxy terminal Gα peptide comprisingcontiguous amino acids of a length described herein which is at leastabout 90%, more preferably, at least about 95%, and most preferably, atleast about 99% identical to the amino acid sequence of the carboxyterminal region of a Gα subunit protein, and which includes at least theseven contiguous terminal residues of the α subunit protein. By way offurther example, the invention can include a minigene which encodes apeptide having an amino acid sequence which is at least about 90%, morepreferably, at least about 95%, and even more preferably, at least about99% identical to the amino acid sequence of the carboxy terminal regionof a Gα subunit protein, and including at least the seven contiguousterminal amino acid residues of a carboxy terminal Gα peptide disclosedherein (SEQ ID Nos: 15-29 and 39-47).

[0102] A minigene described herein is assembled using ordinary molecularbiology techniques, such as those described, for example, in Sambrook etal. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York), in Ausubel et al. (1997, Current Protocols inMolecular Biology, John Wiley & Sons, New York), and in Gerhardt et al.eds. (1994, Methods for General and Molecular Bacteriology, AmericanSociety for Microbiology, Washington, D.C.). Accordingly, the design ofminigene functional elements such as a stop codon and any appropriaterestriction endonuclease sites, is understood to be within the abilityof one skilled in the art of molecular biology. Upon reading the presentdisclosure and examining the particulars of FIG. 1 provided herein, itis a simple matter for the skilled artisan to construct a minigene suchthat it can be useful for the methods of the present invention. Bysimply substituting a desired minigene sequence in place of the minigenesequence shown in FIG. 1, it is a simple matter to construct a minigeneencoding any desired Gα carboxy terminal peptide.

[0103] In various embodiments, the invention includes a vectorcomprising a minigene of the invention. The vector can be used tointroduce a minigene encoding a carboxy terminal Gα peptide into a cell.Any type of vector known in the art is suitable for this purpose,including without limitation, plasmid based vectors, viral basedvectors, and non-DNA vectors. Examples of suitable plasmid based vectorsinclude, without limitation, any plasmid which comprises sequencescapable of facilitating either of propagation and expression of thedesired gene in a prokaryotic or eukaryotic cell. Examples of suitableviral vectors include, but are not limited to, retroviral vectors,adenoviral vectors, and adeno-associated viral vectors. Examples ofnon-DNA vectors include, without limitation, polylysine compounds,liposomes, and the like. In preferred embodiments, the vector comprisinga minigene of the invention is an expression vector.

[0104] The invention additionally includes modified carboxy terminal Gαpeptides having one of the following general formulas: MGX, MZX, or MX,wherein M is a methionine amino acid residue, G is a glycine amino acidresidue, Z is an amino acid residue other than glycine, and X is acarboxy terminal Go peptide sequence. By way of example, a modifiedcarboxy terminal Gα peptide can comprise any of the amino acid sequencesdepicted in FIG. 2B (SEQ ID NOs: 15-29). By way of further example, amodified carboxy terminal Gα peptide of the invention can have thegeneral formula MAX or MX, wherein A is an alanine amino acid residueand X is an amino acid sequence, such as any of SEQ ID Nos 39-47. Inthis embodiment, the modified carboxy terminal Gα peptide of theinvention can comprise an amino acid sequence of the carboxy terminus ofany Gα subunit which is known or becomes known, and can be any suchpeptide which exhibits the property of binding a G protein coupledreceptor and is useful in the methods of the invention.

[0105] In preferred embodiments, modified carboxy terminal Gα peptidesof the invention comprise from at least about three contiguous aminoacids to at least about 54 contiguous amino acids, from at least aboutthree contiguous amino acids to at least about eleven contiguous aminoacids, and at least about eleven contiguous amino acids. In a mostpreferred embodiment, a carboxy terminal Gα peptide comprises an aminoacid sequence which is homologous to the amino acid sequence of at leastthe seven contiguous terminal amino acid residues of the carboxyterminal region of a Gα subunit protein. By way of example, theinvention includes a peptide such as SEQ ID NO: 15 which comprisesthirteen contiguous amino acids, within which is the 5′ modification ofa methionine amino acid residue and a glycine amino acid residue, andthe seven contiguous terminal residues, -G-L-F-, of the 3′ end (i.e. thecarboxy terminus of the peptide) which are homologous to the sevencontiguous terminal amino acid residues of the a subunit of the Gprotein, transducin (Gα_(t)). By way of further example, a modifiedcarboxy terminal Gα peptide of the invention can be any of the peptidesdepicted in FIG. 2B (SEQ ID NOs: 15-29).

[0106] The invention further encompasses a peptide corresponding to thecarboxy terminal region of any Gα subunit which is known or becomesknown, and which exhibits the property of binding a G protein coupledreceptor. In preferred embodiments, such carboxy terminal Gα peptidescomprise from at least about three contiguous amino acids to at leastabout 54 contiguous amino acids, from at least about three contiguousamino acids to at least about eleven contiguous amino acids, and atleast about eleven contiguous amino acids. In a most preferredembodiment, a carboxy terminal Gα peptide of the invention comprises anamino acid sequence which is homologous to the amino acid sequence of atleast the seven contiguous terminal residues of the carboxy terminalregion of a Gα subunit protein. By way of example, a peptide of theinvention can be any of the peptides depicted in FIG. 2D (SEQ ID NOs:39-47).

[0107] A carboxy terminal Gα peptide of the invention should not beconstrued to be limited solely to encoding a carboxy terminal Gα peptidedisclosed herein. Rather, the invention should be construed to includeany peptide comprising an amino acid sequence which shares substantialhomology with the carboxy terminal amino acid sequence of a Gα subunitprotein which is known or becomes known. For example, the invention caninclude a peptide comprising contiguous amino acids of a lengthdescribed herein which is at least-about 90%, more preferably, at leastabout 95%, and even more preferably, at least about 99% identical to theamino acid sequence of the carboxy terminal region of a Gα subunitprotein including at least the seven contiguous terminal amino acidresidues of the a subunit protein. By way of further example, theinvention can include a minigene which encodes a carboxy terminal Gαpeptide or modified carboxy terminal Gα peptide having an amino acidsequence which is at least about 90%, more preferably, at least about95%, and even more preferably, at least about 99% identical to the aminoacid sequence of a carboxy terminal Gα peptide or modified carboxyterminal Gα peptide disclosed herein (SEQ ID Nos: 15-29 and 39-47).

[0108] The invention encompasses any carboxy terminal Gα peptide ormodified carboxy terminal Gα peptide which is encoded by a nucleic acid,such as, a minigene, as well as a carboxy terminal Gα peptide ormodified carboxy terminal Gα peptide which is generated synthetically orbiosynthetically. Any method of peptide synthesis or biosynthesisavailable to a skilled artisan can be employed in preparing peptidesuseful for the methods described herein.

[0109] In alternative embodiments, a Gα carboxy terminal peptide, suchas any of SEQ ID Nos: 30-39, can be useful for the methods describedherein.

[0110] The invention includes a cell comprising a minigene, a modifiedcarboxy terminal Gα peptide, or a carboxy terminal Gα peptide of theinvention. Such a cell can be prokaryotic or eukaryotic. For example,the cell can be a yeast cell, a bacterial cell, an insect cell, axenopus cell, a drosophila cell, or a mammalian cell. In addition, thecell having a minigene introduced therein, can be one which resides in amammal (i.e. in vivo), or alternatively, can be a cell in culture (i.e.in vitro).

[0111] The invention also encompasses pharmaceutical compositions usefulin the methods of the invention. Such compositions included in thepresent invention are now described.

[0112] The invention encompasses the preparation and use ofpharmaceutical compositions comprising one of a minigene encoding a Gαcarboxy terminal peptide, a peptide encoded thereby, and a Gα carboxyterminal peptide otherwise generated as an active ingredient. Such apharmaceutical composition may consist of the active ingredient alone,in a form suitable for administration to a subject, or thepharmaceutical composition may comprise the active ingredient and one ormore pharmaceutically acceptable carriers, one or more additionalingredients, or some combination of these. The active ingredient may bepresent in the pharmaceutical composition in the form of aphysiologically acceptable ester or salt, such as in combination with aphysiologically acceptable cation or anion, as is well known in the art.

[0113] As used herein, the term “pharmaceutically acceptable carrier”means a chemical composition with which the active ingredient may becombined and which, following the combination, can be used to administerthe active ingredient to a subject.

[0114] As used herein, the term “physiologically acceptable” ester orsalt means an ester or salt form of the active ingredient which iscompatible with any other ingredients of the pharmaceutical composition,which is not deleterious to the subject to which the composition is tobe administered.

[0115] The formulations of the pharmaceutical compositions describedherein may be prepared by any method known or hereafter developed in theart of pharmacology. In general, such preparatory methods include thestep of bringing the active ingredient into association with a carrieror one or more other accessory ingredients, and then, if necessary ordesirable, shaping or packaging the product into a desired single- ormulti-dose unit.

[0116] Although the descriptions of pharmaceutical compositions providedherein are principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs,birds including commercially relevant birds such as chickens, ducks,geese, and turkeys.

[0117] Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, intrathecal or another route of administration.Other contemplated formulations include projected nanoparticles,liposomal preparations, resealed erythrocytes containing the activeingredient, and immunologically-based formulations.

[0118] A pharmaceutical composition of the invention may be prepared,packaged, or sold in bulk, as a single unit dose, or as a plurality ofsingle unit doses. As used herein, a “unit dose” is discrete amount ofthe pharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

[0119] The relative amounts of the active ingredient, thepharmaceutically acceptable carrier, and any additional ingredients in apharmaceutical composition of the invention will vary, depending uponthe identity, size, and condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.By way of example, the composition may comprise between 0.1% and 100%(w/w) active ingredient.

[0120] In addition to the active ingredient, a pharmaceuticalcomposition of the invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

[0121] Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

[0122] A formulation of a pharmaceutical composition of the inventionsuitable for oral administration may be prepared, packaged, or sold inthe form of a discrete solid dose unit including, but not limited to, atablet, a hard or soft capsule, a cachet, a troche, or a lozenge, eachcontaining a predetermined amount of the active ingredient. Otherformulations suitable for oral administration include, but are notlimited to, a powdered or granular formulation, an aqueous or oilysuspension, an aqueous or oily solution, or an emulsion.

[0123] As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

[0124] A tablet comprising the active ingredient may, for example, bemade by compressing or molding the active ingredient, optionally withone or more additional ingredients. Compressed tablets may be preparedby compressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

[0125] Tablets may be non-coated or they may be coated using knownmethods to achieve delayed disintegration in the gastrointestinal tractof a subject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

[0126] Hard capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such hardcapsules comprise, the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

[0127] Soft gelatin capsules comprising the active ingredient may bemade using a physiologically degradable composition, such as gelatin.Such soft capsules comprise the active ingredient, which may be mixedwith water or an oil medium such as peanut oil, liquid paraffin, orolive oil.

[0128] Liquid formulations of a pharmaceutical composition of theinvention which are suitable for oral administration may be prepared,packaged, and sold either in liquid form or in the form of a dry productintended for reconstitution with water or another suitable vehicle priorto use.

[0129] Liquid suspensions may be prepared using conventional methods toachieve suspension of the active ingredient in an aqueous or oilyvehicle. Aqueous vehicles include, for example, water and isotonicsaline. Oily vehicles include, for example, almond oil, oily esters,ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconutoil, fractionated vegetable oils, and mineral oils such as liquidparaffin. Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or N-propyl-p-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

[0130] Liquid solutions of the active ingredient in aqueous or oilysolvents may be prepared in substantially the same manner as liquidsuspensions, the primary difference being that the active ingredient isdissolved, rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

[0131] Powdered and granular formulations of a pharmaceuticalpreparation of the invention may be prepared using known methods. Suchformulations may be administered directly to a subject, used, forexample, to form tablets, to fill capsules, or to prepare an aqueous oroily suspension or solution by addition of an aqueous or oily vehiclethereto. Each of these formulations may further comprise one or more ofdispersing or wetting agent, a suspending agent, and a preservative.Additional excipients, such as fillers and sweetening, flavoring, orcoloring agents, may also be included in these formulations.

[0132] A pharmaceutical composition of the invention may also beprepared, packaged, or sold in the form of oil-in-water emulsion or awater-in-oil emulsion. The oily phase may be a vegetable oil such asolive or arachis oil, a mineral oil such as liquid paraffin, or acombination of these. Such compositions may further comprise one or moreemulsifying agents such as naturally occurring gums such as gum acaciaor gum tragacanth, naturally-occurring phosphatides such as soybean orlecithin phosphatide, esters or partial esters derived from combinationsof fatty acids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

[0133] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for rectal administration.Such a composition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

[0134] Suppository formulations may be made by combining the activeingredient with a non-irritating pharmaceutically acceptable excipientwhich is solid at ordinary room temperature (i.e. about 20° C.) andwhich is liquid at the rectal temperature of the subject (i.e. about 37°C. in a healthy human). Suitable pharmaceutically acceptable excipientsinclude, but are not limited to, cocoa butter, polyethylene glycols, andvarious glycerides. Suppository formulations may further comprisevarious additional ingredients including, but not limited to,antioxidants and preservatives.

[0135] Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

[0136] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for vaginal administration.Such a composition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or gel or cream or a solution for vaginalirrigation.

[0137] Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e. such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

[0138] Douche preparations or solutions for vaginal irrigation may bemade by combining the active ingredient with a pharmaceuticallyacceptable liquid carrier. As is well known in the art, douchepreparations may be administered using, and may be packaged within, adelivery device adapted to the vaginal anatomy of the subject. Douchepreparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants, antibiotics, antifungalagents, and preservatives.

[0139] As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrasternal injection, and kidneydialytic infusion techniques.

[0140] Formulations of a pharmaceutical composition suitable forparenteral administration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e. powder or granular) form for reconstitution with asuitable vehicle (e.g. sterile pyrogen-free water) prior to parenteraladministration of the reconstituted composition.

[0141] The pharmaceutical compositions may be prepared, packaged, orsold in the form of a sterile injectable aqueous or oily suspension orsolution. This suspension or solution may be formulated according to theknown art, and may comprise, in addition to the active ingredient,additional ingredients such as the dispersing agents, wetting agents, orsuspending agents described herein. Such sterile injectable formulationsmay be prepared using a non-toxic parenterally-acceptable diluent orsolvent, such as water or 1,3-butane diol, for example. Other acceptablediluents and solvents include, but are not limited to, Ringer'ssolution, isotonic sodium chloride solution, and fixed oils such assynthetic mono- or di-glycerides. Other parentally-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form, in a liposomal preparation, or as acomponent of a biodegradable polymer systems. Compositions for sustainedrelease or implantation may comprise pharmaceutically acceptablepolymeric or hydrophobic materials such as an emulsion, an ion exchangeresin, a sparingly soluble polymer, or a sparingly soluble salt.

[0142] Formulations suitable for topical administration include, but arenot limited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

[0143] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for pulmonary administrationvia the buccal cavity. Such a formulation may comprise dry particleswhich comprise the active ingredient and which have a diameter in therange from about 0.5 to about 7 nanometers, and preferably from about 1to about 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

[0144] Low boiling propellants generally include liquid propellantshaving a boiling point of below 65° F. at atmospheric pressure.Generally the propellant may constitute 50 to 99.9% (w/w) of thecomposition, and the active ingredient may constitute 0.1 to 20% (w/w)of the composition. The propellant may further comprise additionalingredients such as a liquid non-ionic or solid anionic surfactant or asolid diluent (preferably having a particle size of the same order asparticles comprising the active ingredient).

[0145] Pharmaceutical compositions of the invention formulated forpulmonary delivery may also provide the active ingredient in the form ofdroplets of a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

[0146] The formulations described herein as being useful for pulmonarydelivery are also useful for intranasal delivery of a pharmaceuticalcomposition of the invention.

[0147] Another formulation suitable for intranasal administration is acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 to 500 micrometers. Such a formulation isadministered in the manner in which snuff is taken i.e. by rapidinhalation through the nasal passage from a container of the powder heldclose to the nares.

[0148] Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

[0149] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for buccal administration.Such formulations may, for example, be in the form of tablets orlozenges made using conventional methods, and may, for example, 0.1 to20% (w/w) active ingredient, the balance comprising an orallydissolvable or degradable composition and, optionally, one or more ofthe additional ingredients described herein. Alternately, formulationssuitable for buccal administration may comprise a powder or anaerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or aerosolized formulations,when dispersed, preferably have an average particle or droplet size inthe range from about 0.1 to about 200 nanometers, and may furthercomprise one or more of the additional ingredients described herein.

[0150] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for ophthalmicadministration. Such formulations may, for example, be in the form ofeye drops including, for example, a 0.1-1.0% (w/w) solution orsuspension of the active ingredient in an aqueous or oily liquidcarrier. Such drops may further comprise buffering agents, salts, or oneor more other of the additional ingredients described herein. Otherophthalmalmically-administrable formulations which are useful includethose which comprise the active ingredient in microcrystalline form orin a liposomal preparation.

[0151] As used herein, “additional ingredients” include, but are notlimited to, one or more of the following: excipients; surface activeagents; dispersing agents; inert diluents; granulating anddisintegrating agents; binding agents; lubricating agents; sweeteningagents; flavoring agents; coloring agents; preservatives;physiologically degradable compositions such as gelatin; aqueousvehicles and solvents; oily vehicles and solvents; suspending agents;dispersing or wetting agents; emulsifying agents, demulcents; buffers;salts; thickening agents; fillers; emulsifying agents; antioxidants;antibiotics; antifungal agents; stabilizing agents; and pharmaceuticallyacceptable polymeric or hydrophobic materials. Other “additionalingredients” which may be included in the pharmaceutical compositions ofthe invention are known in the art and described, for example in Genaro,ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa., which is incorporated herein by reference.

[0152] Typically dosages of the compound of the invention which may beadministered to an animal, preferably a human, range in amount from 1 μgto about 100 g per killogram of body weight of the animal. While theprecise dosage administered will vary depending upon any number offactors, including but not limited to, the type of animal and type ofdisease state being treated, the age of the animal and the route ofadministration. Preferably, the dosage of the compound will vary fromabout 1 mg to about 10 g per killogram of body weight of the animal.More preferably, the dosage will vary from about 10 mg to about 1 g perkillogram of body weight of the animal.

[0153] The compound may be administered to an animal as frequently asseveral times daily, or it may be administered less frequently, such asonce a day, once a week, once every two weeks, once a month, or evenless frequently, such as once every several months or even once a yearor less. The frequency of the dose will be readily apparent to theskilled artisan and will depend upon any number of factors, including,but not limited to, the type and severity of the disease being treated,the type and age of the animal, etc.

[0154] Methods of using any of a minigene encoding a modified carboxyterminal Gα peptide, a modified peptide encoded thereby, and a modifiedcarboxy terminal Gα peptide otherwise generated, are now described.

[0155] The carboxy terminal peptide of a Gα subunit can be used toinhibit any reaction that requires signaling by a G protein coupledreceptor (GPCR). Known examples of GPCRs for which such inhibition couldbe useful include, without limitation, a wide range of biologicallyactive receptors, such as hormone receptors, viral receptors, growthfactor receptors, chemokine receptors, sensory receptors, andneuroreceptors.

[0156] Accordingly, the invention includes a method of using a modifiedcarboxy terminal Gα peptide to inhibit a G protein-mediated signalingevent in a cell. This method comprises administering to a cell anisolated nucleic acid comprising a minigene which encodes a modified Gαcarboxy terminal peptide. Alternatively, the method comprisesadministering to the cell a modified Gα carboxy terminal peptidedescribed herein, having the property of binding a G protein coupledreceptor. By way of example, the method of inhibiting a Gprotein-mediated signaling event in a cell can include administering tothe cell any of the minigenes, SEQ ID NOs 1-14, or any of the peptides,SEQ ID NOs 15-29 and 39-47. This method can optionally be useful forinhibiting G protein-mediated signaling events in a cell associated withconstitutively activated GPCRs, and antibody-crosslinked irreversiblyactivated GPCRs.

[0157] The invention also includes a method of blocking the site ofinteraction between a G protein and a G protein coupled receptor in acell. This method comprises administering to a cell either of a minigeneor a modified carboxy terminal Gα peptide, such that the modifiedcarboxy terminal Gα peptide, either administered to the cell orexpressed from an administered minigene, blocks the site of interactionbetween the G protein and the G protein coupled receptor in the cell.

[0158] Additionally, the present invention includes a method of using aminigene or modified carboxy terminal Gα peptide encoded thereby toidentify which G protein specifically binds a particular GPCR. Thismethod is useful for identifying which family of G proteins (i.e. G_(i),G_(s), G_(t), G_(q), etc.) specifically binds a known or newlydiscovered GPCR whose G protein partner is not known (i.e. an orphanreceptor). Examples of such GPCRs include, but are not limited to, TSHreceptors, which are important in Graves Disease, and proteinaseactivated receptors (PARs). This method comprises administering to thecell a minigene of the invention or a modified carboxy terminal Gαpeptide encoded thereby, and assessing the level of occurrence of a Gprotein-mediated signaling event associated with either the particularGPCR or a particular G protein. The assessment can be performed asdescribed herein, or in any other manner which is useful in the methodsof the invention.

[0159] The method of identifying which G protein binds a particular GPCRin a cell further comprises comparing the level of occurrence of asignaling event associated with the GPCR in a cell to which a minigeneor a modified carboxy terminal Gα peptide of the invention has beenadministered, with the level of occurrence of that signaling event in acomparable cell to which the minigene or peptide has not beenadministered. In this embodiment, a measurable reduction in the level ofoccurrence of the signaling event in the cell to which the minigene ormodified peptide has been administered compared with the level ofoccurrence of that signaling event in a cell to which the minigene ormodified peptide has not been administered, is an indication that themodified carboxy terminal Gα peptide expressed from the minigene oradministered to the cell as a peptide, binds the particular GPCRinvolved in the signaling event.

[0160] A modified carboxy terminal Gα peptide which is useful in themethod described above can comprise an amino acid sequence which ishomologous with at least the seven contiguous terminal amino acidresidues of a Gα subunit protein.

[0161] Accordingly, a modified carboxy terminal Gα peptide that exhibitsthe ability to bind a particular GPCR and decrease the level ofoccurrence of a signaling event in which the GPCR is involved,identifies the G protein with which the Gα carboxy terminal peptideshares amino acid sequence homology as being the G protein thatspecifically binds the particular GPCR involved in the signaling event.

[0162] In other embodiments, the invention includes methods ofinhibiting in a cell one or more of migration, permeability andproliferation. In these embodiments, the method comprises administeringto the cell either a minigene or a modified carboxy terminal Gα peptide,thereby inhibiting migration, permeability, proliferation, or acombination thereof in a cell.

[0163] The methods described herein can further comprise assessing thelevel of occurrence of a particular signaling event in the cellfollowing the administration of a minigene or a modified carboxyterminal Gα peptide. This assessment of a signaling event can, forexample, include pretreatment of the cell with one or more stimulatingagents (i.e. ligands which bind GPCRs, thereby inducing interaction withtheir cognate G proteins), and measuring downstream events which are theconsequence of a signaling event to be assessed. By way of example, thelevel of occurrence of a signaling event involving the GPCR, PAR-1, canbe assessed by treating the cell with thrombin, which stimulates PAR-1,and measuring a downstream event that results from signaling by PAR-1,such as an accumulation in the cell of inositol phosphate or an increaseof mitogen-activated protein kinase activity in the cell.

[0164] The methods described herein include administering an isolatednucleic acid or a composition described herein to a cell which can be aprokaryotic cell or a eukaryotic cell, and which can be in culture or inthe body of a mammal. Examples of the type of cell to which a minigeneor modified peptide of the invention can be administered in the abovemethods include, but are not limited to, a mammalian cell, such as ahuman cell, and specific cell types thereof, such as an endothelialcell, a neuronal cell, a myocardial cell, and the like.

[0165] In reference to the methods of the invention, protocols which canbe useful for administering to a cell either an isolated nucleic acid ora peptide composition, and which are not described herein are well knownand within the competence of one of ordinary skill in the art of cellculture.

[0166] The present invention additionally relates to methods whichinvolve administering a minigene or modified peptide described herein toa mammal. In reference to these methods, a minigene, a modified peptide,or a pharmaceutical composition comprising either a minigene or modifiedpeptide described herein can be administered to a mammal, preferably ahuman. The relevant pharmacological factors involved in administeringthese agents are either described in previous sections or are well knownin the art and within the competence of a skilled artisan.

[0167] G protein-mediated signaling events are associated with or havebeen implicated as being responsible for many pathological disorders inanimals and humans. For example, aberrant G protein signaling can beinvolved in the pathology associated with processes involvinginflammation, various infections, disease processes, such as thoseaffecting the cardiopulmonary, endocrine, reproductive, digestive,nervous, and immune systems, and connective and vascular tissues, andsurgical complications, such as those following vascular surgery,coronary bypass surgery, abdominal surgery, and organ transplantsurgery. However, methods of treating such disorders have not beenapparent until the present invention.

[0168] Accordingly, the invention includes a method of treating apathological disorder in a mammal. This method can comprise, forexample, administering to the mammal any of a minigene encoding a Gαcarboxy terminal peptide which is known or becomes known, and a modifiedpeptide encoded thereby or otherwise produced, capable of blocking a Gprotein mediated signaling event associated with the pathologicaldisorder. Preferably, a minigene or peptide of the invention isadministered in an amount sufficient to inhibit the G protein-mediatedsignaling event associated with the pathological disorder. Alsopreferred is administration of either a minigene or peptide of theinvention which results in the alleviation of at least one symptom ofthe pathological disorder.

[0169] The invention encompasses a method of treating a condition forwhich an association with a G protein mediated signaling event is knownor becomes known. By way of example, an orphan receptor for which a Gprotein partner is identified, and with which a pathological disorderbecomes associated, can be blocked according to the method described totreat the associated pathological condition.

[0170] Examples of pathological disorders which can be treated using themethod described herein include, but are not limited to, stroke,myocardial infarction, restenosis, atherosclerosis, hypotension,hypertension, angina pectoris, acute heart failure, cardiomyocyteapoptosis, cancers, bacterial infections, fungal infections, protozoaninfections, viral infections, septic shock, pain, chronic allergicdisorders, asthma, inflammatory bowel disease, osteoporosis, rheumatoidarthritis, Grave's disease, post-operative ileus, diabetes, adultrespiratory distress syndrome, myastenia gravis, cardiovascular disease,congestive heart failure, Chagas disease, disorders associated withsolid organ transplant, vascular sclerosis, chronic rejection, chronicobstructive pulmonary disease, urinary retention, testotoxicosis,infertility, ulcers, obesity, benign prostatic hypertrophy, anxiety,epilepsy, schizophrenia, manic depression, Parkinson's disease,Alzheimer's disease, delirium, dementia, drug addiction, anorexia, andbulimia.

[0171] G protein mediated signaling events have been associated withvarious reproductive functions in mammals (Flanagan et al., 1997, Rev.Reprod. 2:113-120; Gromoll et al., 1996, Mol. Cell Endocrinol.125:177-82; Inanobe et al., 1999, J. Physiol. (Lond) 521:19-30).However, methods of modulating reproduction by manipulating G proteinmediated signaling have not been apparent until the present invention.

[0172] Accordingly, the present invention includes a method ofpreventing conception in a mammal. This method comprises administeringto a female either of an isolated nucleic acid comprising a minigenewhich encodes a modified carboxy terminal Gα peptide, or a modifiedcarboxy terminal Gα peptide, thereby preventing conception in the femalemammal. The administration of such a contraceptive can be carried outusing any methods described in previous sections or any well knownmethods which are within the competence of one skilled in the art.

[0173] The invention is now described with reference to the followingExamples. These Examples are provided for the purpose of illustrationonly and the invention is not limited to these Examples, but ratherencompasses all variations which are evident as a result of the teachingprovided herein.

EXAMPLES

[0174] The experiments presented in the following Examples illustratethe construction of a minigene of the invention and the use of selectedminigenes to inhibit G protein-mediated signaling events.

Example 1 Construction of Minigenes

[0175] Construction of the minigenes was performed using standardreagents and molecular cloning techniques. The functional organizationof the minigenes described in this example is illustrated in FIG. 1. TwoDNA components of the minigenes, insert DNA and vector DNA, wereprepared separately.

[0176] Insert DNA was prepared by first annealing complementaryoligonucleotides comprising, from 5′ to 3′, a BamH1 restrictionendonuclease site, a ribosome-binding nucleotide sequence, a methioninecodon for initiating translation of the subsequent peptide sequence, aglycine codon for stabilization of the translated peptide, a nucleotidesequence encoding a Gα carboxy terminal peptide, a stop codon fortranslation termination, and a Hind III restriction endonucleaserestriction site. The 56 bp oligonucleotides were ordered from GreatAmerican Gene Company (Ramona, Calif.) with their 5′ endsphosphorylated. DNA oligonucleotides were annealed by placing them at85° C. for 5 minutes before allowing them to cool to room temperatureslowly. Examples of insert DNA nucleotide sequences are depicted in FIG.2.

[0177] Vector DNA was prepared by restriction endonuclease digestion ofthe commercially available pcDNA 3.1 plasmid vector (Invitrogen;Carlsbad, Calif.) using BamH1 and HindIII restriction endonucleases.After digestion with each restriction enzyme the pcDNA 3.1 plasmidvector was run on an 0.8% agarose gel, the appropriate band cut out, andthe DNA purified (QIAquick Gel Extraction Kit, QIAGEN, Valencia, Calif.)

[0178] The annealed cDNA oligonucleotides were ligated for 1 hour at RTinto pcDNA 3.1 plasmid vector (Invitrogen; Carlsbad, Calif.) previouslycut with BamHI and Hind111. For the ligation reaction the ratio ofinsert to vector was approximately 25 μM to 50 ng. Respectively.Following ligation, the samples were heated to 65° C. for 5 minutes todeactivate the T4 DNA ligase (New England Biolabs, Beverly, Mass.).Electrocompompetent E. coli. (strain AR 1814) were electroporated in thepresence of the resulting ligated insert-vector DNA.

[0179] Plasmid DNA was recovered from bacteria and purified using theQlAprep Miniprep System (QIAGEN; Valencia, Calif.). The presence of theinsert DNA in the purified plasmid DNA was confirmed by performingrestriction analysis using NcoI restriction endonuclease. Restrictionanalysis consisted of digesting purified plasmid DNA with NcoI usingstandard conditions and reagents, followed by electrophoresis of thedigested DNA through a 1.5% agarose gel matrix.

[0180] The results of restriction analysis of purified plasmid DNA areshown in FIG. 3. Lane 1 in FIG. 3 is a 1-kilobase pair DNA ladder usedas a molecular weight standard. The presence of the insert DNA in thedigested plasmid DNA sample is indicated by a pattern of four DNAfragments, shown in FIG. 3 for minigene plasmids pcDNA-Gα_(i) (lane 3),pcDNA-Gα_(i)R (lane 4), and pcDNA-Gα_(q) (lane 5). The absence of theinsert DNA is indicated by a pattern of three DNA fragments, as seenwith the pcDNA 3.1 plasmid vector in lane 2 of FIG. 3.

Example 2 Transfection and Expression of Minigenes in Cells

[0181] The experiments of this example describe the transfection of HEK293 cells with minigene DNA and the analysis of the expression ofminigene DNA and translation products thereof in transfected cells.

[0182] Transfection of Cells and Analysis of Total Cell RNA fromTransfected Cells

[0183] Transfection of HEK 293 cells with the minigene constructs,pcDNA-Gα_(I)R and pcDNA-Gα_(i), and the control construct, pcDNA 3.1,was carried out using calcium phosphate. Total RNA was isolated fromtransfected cells 48 hours post-transfection using a Qiagen Rneasy Kitand QIAshredder (Qiagen, Valencia, Calif.). The isolated total cell RNAwas used as the template in a reverse transcription-polymerase chainreaction (RT-PCR) with commercially available reagents and methods(Clonetech Advantage RT for PCR kit; Clontech, Palo Alto, Calif.) togenerate the corresponding cDNA. The cDNA was then used as the templatein a standard PCR with primers complementary to the nucleotide sequenceof a portion of the insert DNA described in Example 1. PCR productsobtained using this procedure were analyzed using electrophoresisthrough a 1.5% agarose gel matrix. A typical analysis of this type isdepicted in FIG. 4A. Lane 1 is a 1 kilobase DNA ladder. Lane 2 is a PCRproduct obtained with cells transfected with pcDNA-Gα_(i)R. Lane 3 is aPCR product obtained with cells transfected with pcDNA-Gα_(i). Lane 4 isa PCR product obtained with cells transfected with pcDNA3.1.

[0184] Analysis of Gα Carboxy Terminal Peptide Expression in TransfectedCells

[0185] HEK-293 cells transfected with the minigene constructs,pcDNA-Gα_(i)R and pcDNA-Gα_(i), and the control construct, pcDNA 3.1,were lysed and homogenized 48 hour post-transfection using conventionalmethods. The resulting cytosolic extracts were analyzed using highpressure liquid chromatography (HPLC). Eluent corresponding toindividual peaks observed in HPLC analyses was analyzed by ion massspray analysis. HPLC chromatograms of cytosolic extracts from cellstransfected with the minigene constructs, pcDNA-Gα_(i)R andpcDNA-Gα_(i), and the control construct, pcDNA 3.1, are shown in FIG.4B. The ion mass spray analysis for peak 1 from cells transfected withpcDNA-G_(i), and peak 1 from cells transfected with pcDNA-G_(i)Rindicate the presence of a 1450 dalton peptide, i.e. the expectedmolecular weight for each of the 13 amino acid peptide sequences encodedby the minigenes. A comparable peak is absent in the cytosolic extractfrom cells transfected with the control construct.

[0186] The results of the experiments of this example indicate that, intransiently transfected HEK 293 cells, the minigenes are present, andthe Gα carboxy terminal peptides encoded by the minigenes are expressed.

Example 3 Blockade of G Protein-Mediated Signaling Events by Minigenes

[0187] The experiments of this Example illustrate the use of minigenesas effective and specific blockers of G protein-mediated signalingevents. The general approach of these experiments is as follows.Transient transfection of cells with selected minigene DNA is followedby exposure of transfected cells to a specific G protein coupledreceptor (GPCR) agonist and subsequent measurement of appropriatecellular responses associated with stimulation by the agonist.

[0188] Minigenes selected for these experiments include pcDNA-Gα_(i),pcDNA-Gα_(s), pcDNA-Gα_(q), and the random order minigene, pcDNAGα_(i)R. Also included is the vector DNA, pcDNA 3.1, which does notcontain minigene insert DNA. Preparation of these minigenes was carriedout as described in Example 1.

[0189] Inhibition of Muscarinic M₂ Receptor (M₂ mAchR) Activation ofInwardly Rectifying Potassium Channels (GIRKs)

[0190] G protein regulated GIRK channels modulate electrical activity inexcitable cells. A GIRK channel opens as a consequence of a directinteraction with the Gβγ portion of a G protein. Thus, a whole cellpatch clamp recording of inwardly rectifying K⁺ currents can be used asa measure of G protein activity in single intact cells. In this type ofexperiment, a Gα_(i) carboxyl terminal peptide minigene is being testedfor its ability to inhibit M₂ mAChR activation of inwardly rectifying K⁺currents, that is, the ability to block the interaction between a GIRKchannel, the G protein coupled receptor (GPCR), and its cognate Gprotein, G_(i).

[0191] HEK 293 cells were transiently transfected with plasmid DNAencoding GIRK1/GIRK4 channel proteins and DNA comprising one of theminigenes, pcDNA-Gα_(i), pcDNA-Gα_(s), pcDNA-Gα_(q), and pcDNA-Gα_(i)R.After 48 hours of transfection, the cells were superfused with 1micromolar acetylcholine (Ach). Measurement of whole cell currents todetermine the level of G protein mediated signaling activity in cellswas performed as follows. Membrane currents were recorded under voltageclamp using conventional whole cell-patch techniques. Variables arisingfrom transfection and culture conditions were minimized by performingcontrol minigene transfections (i.e. with pcDNA-Gα_(i)R) in parallelwith pcDNA-Gα_(i), pcDNA-Gα_(s), and pcDNA-Gα_(q) minigenetransfections. For analysis of the data the maximal current density(peak amplitude) of ACh-induced inwardly rectifying K⁺ currents weremeasured at −80 millivolts and compared. Summarized data for the maximumamplitude of ACh evoked currents are shown in FIG. 4 for selectedtransfection conditions.

[0192] As shown in FIG. 4, cells transfected with pcDNA-Gα_(i) DNAexhibited a dramatically impaired response to agonist stimulation of M₂mAchR. The data in FIG. 4A demonstrate that superfusion of the cellswith ACh activated inwardly rectifying K⁺ currents in cells transfectedwith pcDNA-Gα_(i)R DNA but did not activate inwardly rectifying K⁺currents in cells transfected with pcDNA-Gα_(i) DNA. Thus, thepcDNA-Gα_(i) minigene completely blocked the agonist-stimulated GIRK1/4response while the random order minigene, pcDNA-Gα_(i)R, which shouldnot have a specific affinity for M₂ mAchR, did not block theagonist-stimulated GIRK1/4 response.

[0193] The data in FIGS. 4B and 4C demonstrate that theagonist-stimulated GIRK1/4 response, i.e. the activation of inwardlyrectifying K⁺ currents, in cells transfected with either of theminigenes, pcDNA-Gα_(s) and pcDNA-Gα_(q), was not significantlydifferent than the agonist-stimulated GIRK1/4 response of cellstransfected with the control minigene, pCDNA-Gα_(i)R. Therefore, thetransfection of cells with minigene DNA encoding the carboxy terminalpeptides of Gα_(s) and Gα_(q), i.e. peptides derived from G proteinsthat do not normally interact with GIRK channels, does not result in ablockade of the agonist-stimulated GIRK1/4 response in cells.

[0194] The results of these experiments taken together indicate that theGIRK1/4 G protein coupled response is effectively and specificallyblocked by transfection with pCDNA-Gα_(i) minigene DNA (Gilchrist etal., 1999, J. Biol. Chem. 274: 6601-6606).

[0195] Blockade of G protein-mediated Signaling Events Involving thePAR-1 Receptor

[0196] The thrombin receptor, PAR-1, is expressed in endothelial cells,and has been reported to activate multiple G proteins. Because PAR-1interacts with multiple G proteins, there are a number of experimentalendpoints which can be used as measures of PAR-1 signaling events. Theexperiments which follow demonstrate the effect on thrombin-stimulatedPAR-1 signaling of transiently transfecting HMEC with selected minigenesand a control DNA and measuring induction of PAR-1 gene expression,intracellular Ca²⁺ levels, intracellular cAMP levels, MAPK activity,inositol phosphate accumulation, and endothelial cell permeability,proliferation, stress fiber formation, and adhesion.

[0197] a. Blockade Of Thrombin-Stimulated Decreases In cAMPAccumulation.

[0198] Isoproterenol serves as an agonist for the GPCR β-adrenergicreceptor. When stimulated by isoporterenol, β-adrenergic receptorsactivate their cognate proteins (G₅) which subsequently stimulateadenylyl cyclase and lead to significant increases in cellular levels ofcyclic adenosine monophosphate (cAMP). Thrombin receptor (PAR1) is knownto couple to multiple G proteins, including G_(i). The measurement ofcAMP levels as an indicator of thrombin-stimulated PAR-1 signaling wascarried out as follows. HMEC were seeded onto 6-well culture dishes at1×10⁵ cells per well at least 24 hours before transfection. The platedcells were transiently transfected with either the empty vector controlDNA, pcDNA3.1, or one of the minigene DNAs, pcDNA-Gα_(i1/2), andpcDNA-Gα_(i)R, at a concentration of 1 microgram of DNA per well.Transfections were performed using Effectene and the accompanyingprotocols (Qiagen, Valencia, Calif.). At 24 hours post-transfection,cells were treated with tritium-labeled adenine having a specificradioactivity of 3 microcuries per milliliter ([³H]-adenine). 24 hourslater, cells were washed once with a solution comprising 1 millimolarisobutylmethylxantine in serum-free media. Cells were subsequentlytreated with a solution comprising 1 micromolar isoproterenol inserum-free media for 30 min at 37° C. to stimulate cAMP production. Forthrombin inhibition reactions, cells were exposed to either of asolution comprising 50 nanomolar thrombin and a solution comprising 1micromolar quinpirole for at least 15 minutes prior to the treatmentwith the isoproterenol solution. Cell treatments were terminated byaspiration of the treatment solution followed by addition of an ice coldsolution comprising 5% trichloroacetic acid. The assessment of cAMPaccumulation in cells was carried out by separation of the acid-solublenucleotides on ion-exchange columns.

[0199] The results of these experiments are depicted in FIGS. 5 and 13,and are expressed as (cAMP/cAMP+ATP)×1000 for each of cells transfectedwith pcDNA3.1, cells transfected with pcDNA-Gα_(i1/2), and cellstransfected with pcDNA-Gα_(i)R. The isoproterenol-stimulated increase incAMP accumulation was inhibited by thrombin treatment in cellstransfected with either pcDNA 3.1 or pcDNA-Gα_(i)R DNA. In contrast,treatment with thrombin did not inhibit isoproterenol-stimulated cAMPaccumulation in cells transfected with pcDNA-Gα_(i1/2) DNA.

[0200] b. Inhibition of Thrombin-Stimulated Proliferation

[0201] Bovine pulmonary arterial endothelial (BPAE) cells weretransfected with pcDNA3.1 (vector only), pcDNA-Gi, or pcDNA-GiR usingelectroporatoration and allowed to recover for 24 hours. Followingrecovery cells were plated in a 96 well plate at equal density. Cellswere allowed to adhere for 3 hours, followed by stimulation with 100 nMthrombin or carrier alone for 18 hours. To measure proliferation, cellswere labeled with 5-bromo-2′deoxyuridine (BrdU) for 2 hrs and then fixedand denatured using the Biotrak cell proliferation ELISA system(Amersham Pharmacia Biotech; London, UK). After blocking, cells wereincubated with peroxidase-labelled anti-BrdU, washed 3× and incubatedwith substrate. The reaction was stopped with IM sulphuric acid and theOD₄₅₀ was measured. Results shown in FIG. 6 provide data which has beennormalized to a value of 1.0 for unstimulated cells transfected withpcDNA-GiR. Thrombin stimulated proliferation of endothelial cells isthought to be primarily through the Gi pathway. When the pcDNA-Gi_(1/2)minigene was present, absolutely no thrombin-mediated increase inproliferation was observed.

[0202] c. Blockade of Thrombin-Stimulated PAR-1 Gene Induction

[0203] Thrombin treatment of cells induces the expression of the geneencoding its GPCR, PAR-1. This induction is mediated through the PAR-1gene promotor sequence. The PAR-1 gene promotor sequence can be made todirect the expression of a reporter gene such as, the luciferase gene,thereby providing a means of observing the level of PAR-1 gene inductionoccurring in response to thrombin treatment of cells.

[0204] PAR-1 gene induction was demonstrated using a luciferase reporterassay as follows. A PAR-1 promoter sequence comprising 1.82 kilobaseswas inserted upstream of the gene, Firefly (Photinus pyralis) luciferasein a pGL2 vector DNA to generate the plasmid DNA, PCR-1/Luc. The seapansy (Renilla reniformis) luciferase gene operably linked to the HerpesSimplex Virus-Thymidine Kinase promoter (TK) in a pRL vector DNA(TK/pRL) served as an internal control to correct for experimentalvariation. HMEC were co-transfected with 100 nanograms each of areporter DNA, PCR-1/Luc or TK/pRL, and one of pcDNA3.1, pcDNA-Gα_(i), orpcDNA-Gα_(i)R using protocols and reagents described in the previousExample. Twenty four hours post-transfection, HMEC were incubated inserum-free culture media (Dulbecco's Modified Eagles Medium, DMEM) for 2hr at 37° C., following which, thrombin was added to the culture mediaat a concentration of 10 nanomolar. The cells were then incubated at 37°C. for four hours and lysed. For pertussis toxin pretreatmentexperiments, pertussis toxin was added to the cells in culture media ata concentration of 100 nanograms per milliliter, and the cells wereincubated at 37° C. for at least 8 hours prior to treatment withthrombin. For the luciferase assay, a 20 microliter aliquot of the celllysate was treated using protocols and reagents provided with the DualLuciferase Reagent Assay System (Promega, Madison, Wis.).

[0205] The results of the luciferase experiments are depicted in FIG. 7.The level of luciferase activity was higher after thrombin treatment inHMEC transiently transfected with any of the plasmids, PCR-1/Luc,pcDNA3.1, and pcDNA-Gα_(i)R as compared with the level luciferaseactivity in these cells in the absence of thrombin stimulation. Incontrast, the level of luciferase activity did not increase in HMECtransfected with pcDNA-Gα_(i). Pertussis toxin pretreatment of cells,which is known to block G_(i) signaling also blocked the increase inluciferase levels. The results of these luciferase experimentsdemonstrate that the induction of PAR-1 gene expression, which isnormally blocked by pertussis toxin, can also be blocked by transienttransfection with a minigene DNA encoding the Gα_(i) carboxy terminalpeptide. In addition, PAR-1 gene expression is not blocked by either ofthe control or random order minigene, pcDNA-Gα_(i)R.

[0206] d. Blockade of Thrombin-Stimulated Intracellular Ca ² Rise

[0207] HMEC were cotransfected with 1 microgram per well of a plasmidDNA encoding human πH3-CD8 and 1 microgram per well each of the controlDNA, pcDNA 3.1, or pcDNA-Gα_(i), pcDNA-Gα_(q), or pcDNA-Gα_(i)R.Transfected cells were seeded on coverslips in a 24 well plate (i.e. 1coverslip per well) at 48 hours post-transfection. Cells were incubatedat 37° C. on the coverslips for 2 hours, following which approximately2×10⁵ microbeads adsorbed with the CD8 receptor protein were added toeach well. The cells and microbeads were treated with a solutioncomprising 10 micromolar Oregon Green 488 BAPTA-1 and 0.1% PluronicF-127 (Molecular Probes, Eugene, Oreg.), and incubated for 20-30 minutesat 37° C. Excess microbeads were removed by rinsing each well twice witha wash solution. Following the rinses, wash solution was added to eachwell, and the levels of intracellular Ca²⁺ in each well were measured atambient temperature. The fluorescence measurements of intracellular Ca²⁺levels were made using an upright Olympus microscope (BX 50WI) equippedwith Hamamatsu digital video camera and a Dell (Pentium II) computerenhanced with MetaMorph 3.5 software. Analysis of fluorescence intensityin the microscopic images was made using the NIH Image Program. Toestablish basal levels of intracellular Ca²⁺, fluorescence intensity wasrecorded in cells 50 seconds prior to treatment of the cells withthrombin. Following thrombin treatment, fluorescence intensity wasrecorded in cells at 10 second intervals for a period of 1600 seconds.

[0208] The tabular results of these experiments are depicted in FIG. 8.The the fluorescence intensities of recorded images are presented as amean±standard error of the mean (SEM) for at least 15 individuallyrecorded cells from each of the following transfection conditions, humanπH3-CD8+the control DNA, pcDNA 3.1, human πH3-CD8+pcDNA-Gα_(i), humanπH3-CD8+pcDNA-Gα_(q), and human πH3-CD8+pcDNA-Gα_(i)R.

[0209] The results of these experiments indicate that transfection ofthe minigene encoding the Gα_(q) carboxy terminal peptide (pcDNA-Gα_(q))blocks a rise in intracellular Ca²⁺ levels following thrombinstimulation. This is the expected result, since the rise inintracellular Ca²⁺ levels is a downstream consequence of the interactionbetween PAR-1 and the G protein, Gq. Transient transfection of the otherminigene DNAs, pcDNA-Gα_(i), or pcDNA-Gα_(i)R, and the control DNA,pcDNA 3.1, do not block the rise in intracellular Ca²⁺ levels. Thusthese experiments demonstrate that the blockade of thrombin-induced risein Ca²⁺ by transfection of a minigene encoding the carboxy terminalpeptide, Gα_(q), is effective and is also specific for Gα_(q).

[0210] e. Blockade of Thrombin-Stimulated Inositol Phosphate (IP)Accumulation

[0211] Thrombin is an agonist for the GPCR, PAR-1, which in-turninteracts with a G protein to initiate signaling events in a cell.Thrombin-induced IP accumulation is one downstream consequence of thisinteraction. Thus, blockade of an increase in response to thrombintreatment can be used to indicate the blockade of a G protein-mediatedsignaling event.

[0212] The increase in IP accumulation in HMEC in response to thrombinstimulation was carried out as follows. HMEC were seeded into 6-wellplates at 1×10⁵ cells per well. Cells were transiently transfected with1 microgram per well of one of the minigene DNAs, pcDNA-Gα_(i), andpcDNA-Gα_(q), or the control DNA, pcDNA3.1. Cells were reseeded at 24hours post-transfection onto 24-well plates, treated withtritium-labeled myoinositol having a specific radioactivity of 4microcuries per milliliter ([H] myoinositol), and incubated for 24 hoursat 37° C. Prior to stimulation 5 mM LicI was added to cells, andincubated for 1 hour. Stimulation was for 5 minutes with 1 nM humanthrombin after which the medium was aspirated and cells lysed byaddition of ice-cold methanol. Supernatant fractions were loaded ontoAG1-8X Dowex columns and washed first with a solution comprising 40millimolar ammonium formate to remove [³H] inositol, and next, with asolution comprising 2 molar ammonium formate to elute [³H] IP.

[0213] The results of IP accumulation experiments are depicted in FIG.9. The data are expressed as a mean of two independent experiments inwhich each DNA was transfected in triplicate (i.e. 1 micgrogram of agiven DNA in each of three wells). The data for each experiment weregenerated using calculations performed as follows: counts per min[³H]-IP fraction/counts per min [³H]-inositol fraction+counts per min[³H]-IP fraction.

[0214] The IP accumulation experiments demonstrate that transfection ofthe minigene encoding the Gα_(q) carboxy terminal peptide (pcDNA-Gα_(q))either alone or in combination with the minigene encoding the Gα_(i)carboxy terminal peptide (pcDNA-Gα_(i)) blocks the accumulation of IPfollowing thrombin stimulation. This is the expected result, since IPaccumulation is a downstream consequence of the interaction betweenPAR-1 and the G protein, G_(q). Transient transfection of pcDNA-Gα_(i)by itself or the control DNA, pcDNA 3.1, do not block IP accumulation.Thus these experiments demonstrate that blockade of thrombin-induced IPaccumulation by transfection of a minigene encoding the carboxy terminalpeptide, Gα_(q), is effective and specific for Gα_(q).

[0215] f. Blockade of Thrombin-Induced Mitogen-activated Protein Kinase(MAPK) Activity

[0216] HMEC were co-transfected with a plasmid DNA encodinghemagglutinin (HA-MAPK) and one of the plasmid DNAs, pcDNA3.1,pcDNA-Gα_(i), pcDNA-Gα_(q), or pcDNA-Gα_(i)R, in the manner described inthe previous Examples. At 48 hours post-transfection, cells weretransferred to serum-free medium and incubated for 2 hours at 37° C. Fortreatment with thrombin, cells were incubated at 37° C. for 5 minutes ina solution comprising 10 millimolar thrombin. Cells were lysed bytreatment with RIPA buffer according to the manufacturer's protocol. Theresulting cell lysates were mixed with a solution comprising the 12CA5antibody and preswollen Protein A-Sepharose, and subjected toimmunoprecipitation using standard protocols. HA-MAPK activity wasmeasured in the precipitated samples by incorporation of radioactivephosphorous (³²P) into myelin basic protein (MBP), i.e. phosphorylationof MBP by HA-MAPK in the presence of ³²P-labeled adenosine triphosphate(ATP). Phosphorylation experiments were performed using standardprotocols and reagents.

[0217] The results of HA-MAPK activity experiments are depicted in FIG.10. The data for HA-MAPK activity are presented as the ³²P incorporationinto MBP per nanomole of protein per minute of the phosphorylationreaction. Measurements of HA-MAPK activity were made with cell lysatesfrom each of the following transfection conditions, HA-MAPK+the controlDNA, pcDNA 3.1, HA-MAPK+pcDNA-Gα_(i), HA-MAPK+pcDN A-Gα_(q), andHA-MAPK+pcDNA-Gα_(i)R.

[0218] The results of these experiments indicate that transfection ofthe minigene encoding the Gα_(q) carboxy terminal peptide (pcDNA-Gα_(q))blocks the increase in MAPK activity levels following thrombinstimulation. This is the expected result, since MAPK activity is inducedas a downstream consequence of the interaction between PAR-1 and the Gprotein, G_(q). Transient transfection of the other minigene DNAs,pcDNA-Gα_(i), or pcDNA-Gα_(i)R, and the control DNA, pcDNA 3.1, do notblock the induction of MAPK activity by thrombin stimulation. Thus theseexperiments demonstrate an effective and specific blockade of a Gprotein-mediated by transfection of a minigene encoding the carboxyterminal peptide, Gα_(q).

[0219] g. Inhibition of Thrombin-Mediated Stress Fiber Formation

[0220] HMEC were seeded on gelatin-coated coverslips, and transientlytransfected with pcDNA (control), or the pcDNA-G12, or pcDNA-G13minigenes in the presence of green fluorescent probe (GFP) expressionvector (cDNAs ratio 5:1). Twenty-four hours later, cells wereserum-starved for an additional 24 hours and then challenged with 10 nMthrombin for 5 min. Cells were washed with phosphate buffer saline(PBS), fixed with 4% paraformaldehyde, and permeabilized with 0.1%Triton X-100. Thereafter, cells were incubated for 30 min at roomtemperature with 1 μM rhodamine-phalloidin (Molecular Probes; Eugene,Oreg.) to visualize polymerized F-actin. Cells were extensively washed,mounted using Vectashield antifade mounting medium (Vector Laboratories,Inc.; Burlingame, Calif.). Cells were observed with an invertedmicroscope (Diaphot 200, Nikon, Inc.; Melville, N.Y.) equipped for bothdifferential interference contrast (DIC) microscopy and epifluorescenceobservation using a 60× oil-immersion objective. Fluorescence and DICimages were recorded for each cell field with a cooled, integrating CCDarray camera (Imagepoint, Photometrix, Ltd.; Manchester, Conn.)connected to the microscope. In each coverslip, 100 GFP-expressing cellswere analyzed. FIG. 11 shows that control cells which are transfectedwith the empty vector (pcDNA 3.1) and are serum starved and unstimulatedhave very few stress fibers (FIG. 11A; top left). Following stimulationwith thrombin stress fibers are quickly induced (FIG. 11B; top right).However if cells are transfected with pcDNA-Gα₁₂ minigene (FIG. 11C;bottom left) or pcDNA-Gα 1₃ minigenes (FIG. 11D; bottom right) thrombindoes not induce stress fiber formation.

[0221] h. Decrease in Thrombin-Induced Adhesion of HL-60 Cells

[0222] HMEC cells were transfected in a 6 well plate using the EffecteneTransfection Reagent (Qiagen; Carlsbad, Calif.) and pcDNA3.1,pcDNA3.1-GiR, pcDNA3.1-G12, or pcDNA3.1-G13. After 24 hrs cells weresplit into a 96 well to obtain 1×10⁴ cells/well. Cells were allowed toadhere for 24 hrs, then stimulated with 1 nM thrombin for 1 hr. Duringthis incubation period 1×10⁵ HL60 cells were labeled with 5 μM calceinacetoxymethyl ester (calcein AM) (Molecular Probes; Eugene, Oreg.) for30 min. Calcein AM labeled HL60 cells were washed twice and added insuspension to the transfected thrombin stimulated HMEC cells. After 1 hrat 37° C. nonadherent HL60 cells were removed by washing and 200 μl ofPBS was added to each well. Fluorescence of the remaining calcein AMlabeled HL60 cells was measured using a fluorescein filter withabsorbance at 494 nm and emission at 517 nm. The results shown in FIG.12 represent data that have been normalized to a value of 1.0 forunstimulated pcDNA-Gα_(i)R transfected cells. The presence of either thepcDNA-Gα₁₂ or pcDNA-Gα₁₃ minigene inhibits the normally observedthrombin-mediated increase in HL-60 cell adhesion to endothelial cells.

[0223] i. Blockade of Thrombin-Induced Transendothelial ElectricalResistance (TEER).

[0224] Endothelial cells undergo morphological changes in response tothrombin stimulation. Such changes coincide with other biochemicalsequelae and with increased proliferation of the endothelial cells. Oneconsequence of the shape change is decreased resistance of the cells toelectrical current. Thus, a measure of the decrease in the resistance ofendothelial cells to electrical current, i.e. transendothelialelectrical resistance (TEER), can be used to measure the occurrence of aG protein-mediated signaling event responsible for the shape change.

[0225] The TEER of HMEC was measured as follows. HMEC were transientlytransfected with 1 microgram per well of one of the minigene DNAs,pcDNA-Gα_(i), and pcDNA-Gα_(s), pcDNA-Gα_(i)R or the control DNA,pcDNA3.1. At 24 hours post-transfection, the cells were reseeded andgrown in culture medium at 37° C. The resulting monolayers of cells weresubjected to an alternating current of 50 microamperes by passing thecurrent at 2 pulses per minute across each monolayer. Treatment withthrombin was carried out as described in the previous Examples.

[0226] The results of these experiments are depicted in FIG. 14. Thebasal level of TEER was decreased by 40% in HMEC transfected withpcDNA-Gα_(s) minigene compared with the control DNA (pcDNA 3.1).Following thrombin stimulation, HMEC transfected with pcDNA 3.1 andpcDNA-Gα_(i)R, exhibited decreases in TEER of 36% and 39%, respectively.In contrast, HMEC transfected with pcDNA-Gα_(s), exhibited a 23%decrease in TEER.

[0227] The results of the TEER experiments demonstrate that Gs mediatessignaling events that ultimately result in endothelial cell shapechanges, and that such pathways can be blocked effectively andspecifically by the pcDNA-Gα_(s) minigene.

[0228] The experiments described in the Examples presented hereindemonstrate that minigenes encoding Gα carboxy terminal peptides areuseful for effectively and specifically targeting the Gα-GPCR interfaceand blocking G protein-mediated signaling events.

[0229] The disclosure of every patent, patent application, andpublication cited herein is hereby incorporated herein by reference inits entirety.

[0230] While this invention has been disclosed with reference tospecific embodiments, it is apparent that other embodiments andvariations of this invention can be devised by others skilled in the artwithout departing from the true spirit and scope of the invention. Theappended claims include all such embodiments and equivalent variations.

1 47 1 57 DNA Artificial Sequence minigene 1 gatccgccgc caccatggaaatcaaggaaa acctgaagga ctgcggcctc ttctgaa 57 2 57 DNA Artificial Sequenceminigene 2 gatccgccgc caccatggga atcaagaaca acctgaagga ctgcggcctcttctgaa 57 3 57 DNA Artificial Sequence minigene 3 gatccgccgc caccatgggaaacggcatca agtgcctctt caacgacaag ctgtgaa 57 4 57 DNA Artificial Sequenceminigene 4 gatccgccgc caccatggga attaaaaaca acttaaagga atgtggactttattgaa 57 5 57 DNA Artificial Sequence minigene 5 gatccgccgc caccatgggaatcgccaaaa acctgcgggg ctgtggactc tactgaa 57 6 57 DNA Artificial Sequenceminigene 6 gatccgccgc caccatggga attgccaaca acctccgggg ctgcggcttgtactgaa 57 7 57 DNA Artificial Sequence minigene 7 gatccgccgc caccatgggaatacagaaca atctcaagta cattggcctt tgctgaa 57 8 57 DNA Artificial Sequenceminigene 8 gatccgccgc caccatggga ctgcagctga acctgaagga gtacaatctggtctgaa 57 9 57 DNA Artificial Sequence minigene 9 gatccgccgc caccatgggactccagttga acctgaagga gtacaatgca gtctgaa 57 10 57 DNA ArtificialSequence minigene 10 gatccgccgc caccatggga cagcggatgc acctcaagcagtatgagctc ttgtgaa 57 11 57 DNA Artificial Sequence minigene 11gatccgccgc caccatggga ctacagctaa acctaaggga attcaacctt gtctgaa 57 12 57DNA Artificial Sequence minigene 12 gatccgccgc caccatggga ctcgcccggtacctggacga gattaatctg ctgtgaa 57 13 57 DNA Artificial Sequence minigene13 gatccgccgc caccatggga ctgcaggaga acctgaagga catcatgctg cagtgaa 57 1457 DNA Artificial Sequence minigene 14 gatccgccgc caccatggga cagcgcatgcaccttcgtca gtacgagctg ctctgaa 57 15 13 PRT Artificial Sequence G alpha tpeptide 15 Met Gly Ile Lys Glu Asn Leu Lys Asp Cys Gly Leu Phe 1 5 10 1613 PRT Artificial Sequence G alpha i 1/2 peptide 16 Met Gly Ile Lys AsnAsn Leu Lys Asp Cys Gly Leu Phe 1 5 10 17 13 PRT Artificial Sequence Galpha i R peptide 17 Met Gly Asn Gly Ile Lys Cys Leu Phe Asn Asp Lys Leu1 5 10 18 13 PRT Artificial Sequence G alpha i 3 peptide 18 Met Gly IleLys Asn Asn Leu Lys Glu Cys Gly Leu Tyr 1 5 10 19 13 PRT ArtificialSequence G alpha o 2 peptide 19 Met Gly Ile Ala Lys Asn Leu Arg Gly CysGly Leu Tyr 1 5 10 20 13 PRT Artificial Sequence G alpha o 1 peptide 20Met Gly Ile Ala Asn Asn Leu Arg Gly Cys Gly Leu Tyr 1 5 10 21 13 PRTArtificial Sequence G alpha z 21 Met Gly Ile Gln Asn Asn Leu Lys Tyr IleGly Leu Cys 1 5 10 22 13 PRT Artificial Sequence G alpha 11 peptide 22Met Gly Leu Gln Leu Asn Leu Lys Glu Tyr Asn Leu Val 1 5 10 23 13 PRTArtificial Sequence G alpha q peptide 23 Met Gly Leu Gln Leu Asn Leu LysGlu Tyr Asn Ala Val 1 5 10 24 13 PRT Artificial Sequence G alpha olfpeptide 24 Met Gly Gln Arg Met His Leu Lys Gln Tyr Glu Leu Leu 1 5 10 2513 PRT Artificial Sequence G alpha 14 peptide 25 Met Gly Leu Gln Leu AsnLeu Arg Glu Phe Asn Leu Val 1 5 10 26 13 PRT Artificial Sequence G alpha15/16 peptide 26 Met Gly Leu Ala Arg Tyr Leu Asp Glu Ile Asn Leu Leu 1 510 27 13 PRT Artificial Sequence G alpha 12 peptide 27 Met Gly Leu GlnGlu Asn Leu Lys Asp Ile Met Leu Gln 1 5 10 28 13 PRT Artificial SequenceG alpha 13 peptide 28 Met Gly Leu His Asp Asn Leu Lys Gln Leu Met LeuGln 1 5 10 29 13 PRT Artificial Sequence G alpha s peptide 29 Met GlyGln Arg Met His Leu Arg Gln Tyr Glu Leu Leu 1 5 10 30 33 DNA Homosapiens 30 attaaaaaca acttaaagga atgtggactt tat 33 31 33 DNA Homosapiens 31 atcgccaaaa acctgcgggg ctgtggactc tac 33 32 33 DNA Homosapiens 32 atacagaaca atctcaagta cattggcctt tgc 33 33 33 DNA Homosapiens 33 ctgcagctga acctgaagga gtacaatctg gtc 33 34 33 DNA Homosapiens 34 cagcggatgc acctcaagca gtatgagctc ttg 33 35 33 DNA Homosapiens 35 ctacagctaa acctaaggga attcaacctt gtc 33 36 33 DNA Homosapiens 36 ctcgcccggt acctggacga gattaatctg ctg 33 37 33 DNA Homosapiens 37 ctgcaggaga acctgaagga catcatgctg cag 33 38 33 DNA Homosapiens 38 ctgcatgaca acctcaagca gcttatgcta cag 33 39 11 PRT Homosapiens 39 Ile Lys Asn Asn Leu Lys Glu Cys Gly Leu Tyr 1 5 10 40 11 PRTHomo sapiens 40 Ile Ala Lys Asn Leu Arg Gly Cys Gly Leu Tyr 1 5 10 41 11PRT Homo sapiens 41 Ile Gln Asn Asn Leu Lys Tyr Ile Gly Leu Cys 1 5 1042 11 PRT Homo sapiens 42 Leu Gln Leu Asn Leu Lys Glu Tyr Asn Leu Val 15 10 43 11 PRT Homo sapiens 43 Gln Arg Met His Leu Lys Gln Tyr Glu LeuLeu 1 5 10 44 11 PRT Homo sapiens 44 Leu Gln Leu Asn Leu Arg Glu Phe AsnLeu Val 1 5 10 45 11 PRT Homo sapiens 45 Leu Ala Arg Tyr Leu Asp Glu IleAsn Leu Leu 1 5 10 46 11 PRT Homo sapiens 46 Leu Gln Glu Asn Leu Lys AspIle Met Leu Gln 1 5 10 47 11 PRT Homo sapiens 47 Leu His Asp Asn Leu LysGln Leu Met Leu Gln 1 5 10

What is claimed is:
 1. An isolated nucleic acid comprising a minigene,wherein said minigene encodes a modified carboxy terminal Gα peptide,wherein said peptide blocks the site of interaction between a G proteinand a G protein coupled receptor in a cell.
 2. The isolated nucleic acidof claim 1, further compising at least one of a promoter and a ribosomalbinding site.
 3. The isolated nucleic acid of claim 2, furthercomprising a translation initiation codon and a translation terminationcodon.
 4. The isolated nucleic acid of claim 1, wherein said cell is ahuman cell.
 5. The isolated nucleic acid of claim 1, wherein saidmodified carboxy terminal Gα peptide has a general formula selected fromthe group consisting of MGX, MX, and MZX, wherein M is a methionineamino acid residue, wherein G is a glycine amino acid residue, wherein Zis an amino acid residue other than a glycine amino acid residue, andwherein X is a carboxy terminal Gα peptide, which peptide comprises anamino acid sequence of the carboxy terminus of a Gα subunit, whichpeptide has the property of binding a G protein coupled receptor.
 6. Theisolated nucleic acid of claim 5, wherein X comprises from at leastabout three contiguous amino acids to at least about 54 contiguous aminoacids.
 7. The isolated nucleic acid of claim 6, wherein X comprises fromat least about three contiguous amino acids to at least about elevencontiguous amino acids.
 8. The isolated nucleic acid of claim 7, whereinX comprises at least about eleven contiguous amino acids.
 9. Theisolated nucleic acid of claim 5, wherein X comprises the sevencontiguous terminal amino acid residues of the carboxy terminus of saidGα subunit.
 10. The isolated nucleic acid of claim 5, wherein the aminoacid sequence of said modified carboxy terminal Gα peptide is selectedfrom the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, and
 29. 11. The isolated nucleic acid of claim1, wherein the nucleotide sequence of said minigene is selected from thegroup consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,and
 14. 12. A composition comprising a modified carboxy terminal Gαpeptide having a general formula selected from the group consisting ofMGX, MX, and MZX, wherein M is a methionine amino acid residue, whereinG is a glycine amino acid residue, wherein Z is an amino acid residueother than a glycine amino acid residue, and wherein X is a carboxyterminal Gα peptide, which peptide comprises an amino acid sequence ofthe carboxy terminus of a Gα subunit, which peptide has the property ofbinding a G protein coupled receptor.
 13. The composition of claim 12,wherein X comprises from at least about three contiguous amino acids toat least about 54 contiguous amino acids.
 14. The composition of claim13, wherein X comprises from at least about three contiguous amino acidsto at least about eleven contiguous amino acids.
 15. The composition ofclaim 14, wherein X comprises at least about eleven contiguous aminoacids.
 16. The composition of claim 12, wherein X comprises the sevencontiguous terminal amino acid residues of the carboxy terminus of saidGα subunit.
 17. The composition of claim 12, wherein the amino acidsequence of said modified carboxy terminal Gα peptide is selected fromthe group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, and
 29. 18. A pharmaceutical composition comprisingthe isolated nucleic acid of claim 1 and a pharmaceutically acceptablecarrier.
 19. A pharmaceutical composition comprising a modified carboxyterminal Gα peptide of claim 12 and a pharmaceutically acceptablecarrier.
 20. A method of inhibiting a G protein-mediated signaling eventin a cell, said method comprising administering to said cell an isolatednucleic acid comprising a minigene, wherein said minigene encodes amodified carboxy terminal Gα peptide, whereby following saidadministration, said peptide inhibits said G protein mediated signalingevent in said cell.
 21. The method of claim 20, wherein said cell is ahuman cell.
 22. The method of claim 20, wherein said modified carboxyterminal Gα peptide has a general formula selected from the groupconsisting of MGX, MX, and MZX, wherein M is a methionine amino acidresidue, wherein G is a glycine amino acid residue, wherein Z is anamino acid residue other than a glycine amino acid residue, and whereinX is a carboxy terminal Gα peptide, which peptide comprises an aminoacid sequence of the carboxy terminus of a Gα subunit, which peptide hasthe property of binding a G protein coupled receptor.
 23. The method ofclaim 22, wherein X comprises from at least about three contiguous aminoacids to at least about 54 contiguous amino acids.
 24. The method ofclaim 23, wherein X comprises from at least about three contiguous aminoacids to at least about eleven contiguous amino acids.
 25. The method ofclaim 24, wherein X comprises at least about eleven contiguous aminoacids.
 26. The method of claim 22, wherein X comprises the sevencontiguous terminal amino acid residues of the carboxy terminus of saidGα subunit.
 27. The method of claim 22, wherein the amino acid sequenceof said modified carboxy terminal Gα peptide is selected from the groupconsisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, and
 29. 28. The method of claim 20, wherein the nucleotidesequence of said minigene is selected from the group consisting of SEQID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and
 14. 29. A methodof blocking the site of interaction between a G protein and a G proteincoupled receptor in a cell, said method comprising administering to saidcell an isolated nucleic acid comprising a minigene, wherein saidminigene encodes a modified carboxy terminal Gα peptide, wherebyfollowing said administration, said peptide blocks the site ofinteraction between the G protein and the G protein coupled receptor inthe cell.
 30. The method of claim 29, wherein said cell is a human cell.31. The method of claim 29, wherein said modified carboxy terminal Gαpeptide has a general formula selected from the group consisting of MGX,MX, and MZX, wherein M is a methionine amino acid residue, wherein G isa glycine amino acid residue, wherein Z is an amino acid residue otherthan a glycine amino acid residue, and wherein X is a carboxy terminalGα peptide, which peptide comprises an amino acid sequence of thecarboxy terminus of a Gα subunit, which peptide has the property ofbinding a G protein coupled receptor.
 32. The method of claim 31,wherein X comprises from at least about three contiguous amino acids toat least about 54 contiguous amino acids.
 33. The method of claim 32,wherein X comprises from at least about three contiguous amino acids toat least about eleven contiguous amino acids.
 34. The method of claim31, wherein X comprises at least about eleven contiguous amino acids.35. The method of claim 31, wherein X comprises the seven contiguousterminal amino acid residues of the carboxy terminus of said Gα subunit.36. The method of claim 31, wherein the amino acid sequence of saidmodified carboxy terminal Gα peptide is selected from the groupconsisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, and
 29. 37. The method of claim 29, wherein the nucleotidesequence of said minigene is selected from the group consisting of SEQID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and
 14. 38. A methodof identifying which G protein binds a G protein coupled receptor in acell, said method comprising administering to the cell an isolatednucleic acid comprising a minigene, wherein said minigene encodes amodified carboxy terminal Gα peptide, and assessing the level ofoccurrence of a signaling event in said cell, which event is associatedwith said G protein coupled receptor, wherein a measurable reduction inthe level of occurrence of said signaling event in said cell comparedwith the level of occurrence of said signaling event in a cell to whichsaid isolated nucleic acid is not administered, is an indication thatsaid modified carboxy terminal Gα peptide identifies a G protein thatbinds said G protein coupled receptor.
 39. A method of treating apathological disorder in a mammal, said method comprising administeringto said mammal an amount of an isolated nucleic acid comprising aminigene, wherein said minigene encodes a modified carboxy terminal Gαpeptide, and wherein said amount of said isolated nucleic acid issufficient to inhibit a G protein-mediated signaling event associatedwith said pathological disorder, thereby alleviating at least onesymptom of said pathological disorder.
 40. The method of claim 39,wherein said modified carboxy terminal Gα peptide has a general formulaselected from the group consisting of MGX, MX, and MZX, wherein M is amethionine amino acid residue, wherein G is a glycine amino acidresidue, wherein Z is an amino acid residue other than a glycine aminoacid residue, and wherein X is a carboxy terminal Gα peptide, whichpeptide comprises an amino acid sequence of the carboxy terminus of a Gαsubunit, which peptide has the property of binding a G protein coupledreceptor.
 41. The method of claim 40, wherein X comprises from at leastabout three contiguous amino acids to at least about 54 contiguous aminoacids.
 42. The method of claim 41, wherein X comprises from at leastabout three contiguous amino acids to at least about eleven contiguousamino acids.
 43. The method of claim 42, wherein X comprises at leastabout eleven contiguous amino acids.
 44. The method of claim 40, whereinX comprises the seven contiguous terminal amino acid residues of thecarboxy terminus of said Gα subunit.
 45. The method of claim 40, whereinthe amino acid sequence of said modified carboxy terminal Gα peptide isselected from the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, and
 29. 46. The method of claim 39,wherein the nucleotide sequence of said minigene is selected from thegroup consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, and
 14. 47. A method of treating a pathological disorder in amammal, said method comprising administering to said mammal an amount ofthe modified Gα carboxy terminal peptide of claim 5 sufficient toinhibit a G protein-mediated signaling event associated with saidpathological disorder, thereby alleviating at least one symptom of saidpathological disorder.
 48. The method of claim 47, wherein the aminoacid sequence of said modified Gα carboxy terminal peptide is selectedfrom the group consisting of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, and
 29. 49. The method of claim 39, wherein saidpathological disorder is selected from the group consisting of stroke,myocardial infarction, restenosis, atherosclerosis, hypotension,hypertension, angina pectoris, acute heart failure, cardiomyocyteapoptosis, cancers, bacterial infections, fungal infections, protozoaninfections, viral infections, septic shock, pain, chronic allergicdisorders, asthma, inflammatory bowel disease, osteoporosis, rheumatoidarthritis, Grave's disease, post-operative ileus, diabetes, adultrespiratory distress syndrome, myastenia gravis, cardiovascular disease,congestive heart failure, Chagas disease, disorders associated withsolid organ transplant, vascular sclerosis, chronic rejection, chronicobstructive pulmonary disease, urinary retention, testotoxicosis,infertility, ulcers, obesity, benign prostatic hypertrophy, anxiety,epilepsy, schizophrenia, manic depression, Parkinson's disease,Alzheimer's disease, delirium, dementia, drug addiction, anorexia, andbulimia.
 50. A method of inhibiting one or more of migration,permeability, and proliferation of a cell, said method comprisingadministering to said cell an isolated nucleic acid comprising aminigene, wherein said minigene encodes a modified carboxy terminal Gαpeptide, wherein said peptide blocks a G protein-mediated signalingevent in said cell, thereby inhibiting proliferation of said cell.
 51. Amethod of inhibiting one or more of migration, permeability, andproliferation of a cell, said method comprising administering to saidcell a modified carboxy terminal Gα peptide, wherein said peptide blocksa G protein-mediated signaling event in said cell, thereby inhibitingproliferation of said cell.
 52. The method of claim 51, wherein, saidcell is a human cell.
 53. A method of treating a disorder associatedwith an endothelial cell in a mammal, said method comprisingadministering to said endothelial cell of said mammal an isolatednucleic acid comprising a minigene, wherein said minigene encodes amodified carboxy terminal Gα peptide, wherein said peptide blocks a Gprotein-mediated signaling event in said endothelial cell, therebyalleviating at least one symptom of said disorder associated with saidendothelial cell in said mammal.
 54. A method of treating a disorderassociated with an endothelial cell in a mammal, said method comprisingadministering to said endothelial cell of said mammal a modified carboxyterminal Gα peptide of claim 12, wherein said peptide blocks a Gprotein-mediated signaling event in said endothelial cell, therebyalleviating at least one symptom of said disorder associated with saidendothelial cell in said mammal.
 55. A composition comprising a peptideselected from the group consisting of SEQ ID Nos: 39, 40, 41, 42, 43,44, 45, 46, and
 47. 56. A method of preventing conception in a femalemammal, said method comprising administering to said female mammal anisolated nucleic acid comprising a minigene, wherein said minigeneencodes a modified carboxy terminal Gα peptide, thereby preventingconception in said female mammal.
 57. A method of preventing conceptionin a female mammal, said method comprising administering to said femalemammal a modified carboxy terminal Gα peptide of claim 12, therebypreventing conception in said female mammal.